US20130224243A1 - Use of herpes vectors for tumor therapy - Google Patents
Use of herpes vectors for tumor therapy Download PDFInfo
- Publication number
- US20130224243A1 US20130224243A1 US13/769,182 US201313769182A US2013224243A1 US 20130224243 A1 US20130224243 A1 US 20130224243A1 US 201313769182 A US201313769182 A US 201313769182A US 2013224243 A1 US2013224243 A1 US 2013224243A1
- Authority
- US
- United States
- Prior art keywords
- tumor
- cells
- virus
- herpes simplex
- inoculated
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000013598 vector Substances 0.000 title claims abstract description 69
- 206010028980 Neoplasm Diseases 0.000 title claims description 205
- 238000002560 therapeutic procedure Methods 0.000 title description 3
- XQFRJNBWHJMXHO-RRKCRQDMSA-N IDUR Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(I)=C1 XQFRJNBWHJMXHO-RRKCRQDMSA-N 0.000 title description 2
- 230000002950 deficient Effects 0.000 claims abstract description 50
- 206010061289 metastatic neoplasm Diseases 0.000 claims abstract description 27
- 239000002773 nucleotide Substances 0.000 claims abstract description 21
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 21
- 230000009885 systemic effect Effects 0.000 claims abstract description 18
- 230000005975 antitumor immune response Effects 0.000 claims abstract description 16
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 11
- 210000004027 cell Anatomy 0.000 claims description 171
- 241000700584 Simplexvirus Species 0.000 claims description 102
- 241000700605 Viruses Species 0.000 claims description 96
- 210000004881 tumor cell Anatomy 0.000 claims description 65
- 238000000034 method Methods 0.000 claims description 45
- 230000028993 immune response Effects 0.000 claims description 29
- 239000000203 mixture Substances 0.000 claims description 26
- 102000004127 Cytokines Human genes 0.000 claims description 12
- 108090000695 Cytokines Proteins 0.000 claims description 12
- 239000013603 viral vector Substances 0.000 claims description 10
- 230000001900 immune effect Effects 0.000 claims description 9
- 230000001939 inductive effect Effects 0.000 claims description 8
- 201000001441 melanoma Diseases 0.000 claims description 8
- 101150096316 5 gene Proteins 0.000 claims description 7
- 206010009944 Colon cancer Diseases 0.000 claims description 7
- 206010029260 Neuroblastoma Diseases 0.000 claims description 7
- 230000002238 attenuated effect Effects 0.000 claims description 7
- 101150027427 ICP4 gene Proteins 0.000 claims description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 6
- 102000000505 Ribonucleotide Reductases Human genes 0.000 claims description 6
- 108010041388 Ribonucleotide Reductases Proteins 0.000 claims description 6
- 230000010076 replication Effects 0.000 claims description 5
- 206010006187 Breast cancer Diseases 0.000 claims description 4
- 102000019034 Chemokines Human genes 0.000 claims description 4
- 108010012236 Chemokines Proteins 0.000 claims description 4
- 208000029742 colonic neoplasm Diseases 0.000 claims description 4
- 102200033969 rs1555427498 Human genes 0.000 claims description 4
- 206010041823 squamous cell carcinoma Diseases 0.000 claims description 4
- 206010025323 Lymphomas Diseases 0.000 claims description 3
- 208000000172 Medulloblastoma Diseases 0.000 claims description 3
- 241000700618 Vaccinia virus Species 0.000 claims description 3
- 230000001177 retroviral effect Effects 0.000 claims description 3
- 241000701161 unidentified adenovirus Species 0.000 claims description 3
- 229960005486 vaccine Drugs 0.000 claims description 3
- 206010003571 Astrocytoma Diseases 0.000 claims description 2
- 208000026310 Breast neoplasm Diseases 0.000 claims description 2
- 206010014967 Ependymoma Diseases 0.000 claims description 2
- 208000008839 Kidney Neoplasms Diseases 0.000 claims description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 2
- 206010027406 Mesothelioma Diseases 0.000 claims description 2
- 201000004404 Neurofibroma Diseases 0.000 claims description 2
- 201000010133 Oligodendroglioma Diseases 0.000 claims description 2
- 206010033128 Ovarian cancer Diseases 0.000 claims description 2
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 2
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 2
- 206010060862 Prostate cancer Diseases 0.000 claims description 2
- 206010038389 Renal cancer Diseases 0.000 claims description 2
- 208000005017 glioblastoma Diseases 0.000 claims description 2
- 201000010536 head and neck cancer Diseases 0.000 claims description 2
- 208000014829 head and neck neoplasm Diseases 0.000 claims description 2
- 206010073071 hepatocellular carcinoma Diseases 0.000 claims description 2
- 201000010982 kidney cancer Diseases 0.000 claims description 2
- 201000005202 lung cancer Diseases 0.000 claims description 2
- 208000020816 lung neoplasm Diseases 0.000 claims description 2
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 2
- 201000009020 malignant peripheral nerve sheath tumor Diseases 0.000 claims description 2
- 206010027191 meningioma Diseases 0.000 claims description 2
- 208000007538 neurilemmoma Diseases 0.000 claims description 2
- 208000029974 neurofibrosarcoma Diseases 0.000 claims description 2
- 201000002528 pancreatic cancer Diseases 0.000 claims description 2
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 2
- 201000001514 prostate carcinoma Diseases 0.000 claims description 2
- 206010039667 schwannoma Diseases 0.000 claims description 2
- 241000699670 Mus sp. Species 0.000 description 50
- 108010065805 Interleukin-12 Proteins 0.000 description 45
- 101000883798 Homo sapiens Probable ATP-dependent RNA helicase DDX53 Proteins 0.000 description 41
- 102100038236 Probable ATP-dependent RNA helicase DDX53 Human genes 0.000 description 41
- 238000011081 inoculation Methods 0.000 description 41
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 22
- 108091007433 antigens Proteins 0.000 description 21
- 102000036639 antigens Human genes 0.000 description 21
- 239000000427 antigen Substances 0.000 description 20
- 230000002601 intratumoral effect Effects 0.000 description 19
- 230000004614 tumor growth Effects 0.000 description 17
- 230000000259 anti-tumor effect Effects 0.000 description 16
- 210000001744 T-lymphocyte Anatomy 0.000 description 15
- 230000000694 effects Effects 0.000 description 15
- 238000011725 BALB/c mouse Methods 0.000 description 14
- 230000012010 growth Effects 0.000 description 14
- 241000699666 Mus <mouse, genus> Species 0.000 description 13
- 108090000623 proteins and genes Proteins 0.000 description 13
- 238000007920 subcutaneous administration Methods 0.000 description 13
- 241001465754 Metazoa Species 0.000 description 11
- 241001529936 Murinae Species 0.000 description 11
- 230000002146 bilateral effect Effects 0.000 description 11
- 239000013612 plasmid Substances 0.000 description 11
- 239000003981 vehicle Substances 0.000 description 11
- 230000004083 survival effect Effects 0.000 description 10
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 9
- 239000000872 buffer Substances 0.000 description 9
- 108090000765 processed proteins & peptides Proteins 0.000 description 9
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 9
- 239000012636 effector Substances 0.000 description 8
- 208000015181 infectious disease Diseases 0.000 description 8
- 230000005764 inhibitory process Effects 0.000 description 8
- 230000004044 response Effects 0.000 description 8
- 102100037850 Interferon gamma Human genes 0.000 description 7
- 108010074328 Interferon-gamma Proteins 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- 101150066555 lacZ gene Proteins 0.000 description 7
- 210000004988 splenocyte Anatomy 0.000 description 7
- 230000003612 virological effect Effects 0.000 description 7
- 238000013296 A/J mouse Methods 0.000 description 6
- 108091093088 Amplicon Proteins 0.000 description 6
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000013459 approach Methods 0.000 description 6
- 230000009089 cytolysis Effects 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 210000000612 antigen-presenting cell Anatomy 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 238000012546 transfer Methods 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- 210000003501 vero cell Anatomy 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 230000030833 cell death Effects 0.000 description 4
- 230000001472 cytotoxic effect Effects 0.000 description 4
- 230000002519 immonomodulatory effect Effects 0.000 description 4
- 230000006872 improvement Effects 0.000 description 4
- 238000011065 in-situ storage Methods 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 3
- 101800001467 Envelope glycoprotein E2 Proteins 0.000 description 3
- 208000009889 Herpes Simplex Diseases 0.000 description 3
- 241000700588 Human alphaherpesvirus 1 Species 0.000 description 3
- 108010002350 Interleukin-2 Proteins 0.000 description 3
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 3
- 239000012980 RPMI-1640 medium Substances 0.000 description 3
- 101800001271 Surface protein Proteins 0.000 description 3
- 102000006601 Thymidine Kinase Human genes 0.000 description 3
- 108020004440 Thymidine kinase Proteins 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 231100000433 cytotoxic Toxicity 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000003937 drug carrier Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 238000009169 immunotherapy Methods 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 229960004857 mitomycin Drugs 0.000 description 3
- 238000010172 mouse model Methods 0.000 description 3
- 238000011275 oncology therapy Methods 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 238000012809 post-inoculation Methods 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 229960005322 streptomycin Drugs 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000001356 surgical procedure Methods 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 238000011870 unpaired t-test Methods 0.000 description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 2
- 208000003950 B-cell lymphoma Diseases 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 2
- 238000011765 DBA/2 mouse Methods 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 241000710188 Encephalomyocarditis virus Species 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 2
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 description 2
- 102000043129 MHC class I family Human genes 0.000 description 2
- 108091054437 MHC class I family Proteins 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 241001068263 Replication competent viruses Species 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 108020005202 Viral DNA Proteins 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 230000006023 anti-tumor response Effects 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 201000008274 breast adenocarcinoma Diseases 0.000 description 2
- 208000035269 cancer or benign tumor Diseases 0.000 description 2
- 229960004424 carbon dioxide Drugs 0.000 description 2
- 235000011089 carbon dioxide Nutrition 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 201000010897 colon adenocarcinoma Diseases 0.000 description 2
- 201000010989 colorectal carcinoma Diseases 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000000120 cytopathologic effect Effects 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000012645 endogenous antigen Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 230000009036 growth inhibition Effects 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 238000002513 implantation Methods 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 230000002101 lytic effect Effects 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 230000001394 metastastic effect Effects 0.000 description 2
- 210000000822 natural killer cell Anatomy 0.000 description 2
- 230000000683 nonmetastatic effect Effects 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- ZJAOAACCNHFJAH-UHFFFAOYSA-N phosphonoformic acid Chemical compound OC(=O)P(O)(O)=O ZJAOAACCNHFJAH-UHFFFAOYSA-N 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 230000037452 priming Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 210000004176 reticulum cell Anatomy 0.000 description 2
- 238000003118 sandwich ELISA Methods 0.000 description 2
- 230000002269 spontaneous effect Effects 0.000 description 2
- 238000012384 transportation and delivery Methods 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 238000002255 vaccination Methods 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 241000282552 Chlorocebus aethiops Species 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 208000001382 Experimental Melanoma Diseases 0.000 description 1
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 1
- 101100279830 Friend murine leukemia virus env gene Proteins 0.000 description 1
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 1
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 1
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 1
- 101000746373 Homo sapiens Granulocyte-macrophage colony-stimulating factor Proteins 0.000 description 1
- 241000701074 Human alphaherpesvirus 2 Species 0.000 description 1
- 101100195053 Human herpesvirus 1 (strain 17) RIR1 gene Proteins 0.000 description 1
- 108010047761 Interferon-alpha Proteins 0.000 description 1
- 102000006992 Interferon-alpha Human genes 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 108010002386 Interleukin-3 Proteins 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 108010002586 Interleukin-7 Proteins 0.000 description 1
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 description 1
- 101000746372 Mus musculus Granulocyte-macrophage colony-stimulating factor Proteins 0.000 description 1
- CAUBWLYZCDDYEF-UHFFFAOYSA-N N-Nitroso-N-methylurethane Chemical compound CCOC(=O)N(C)N=O CAUBWLYZCDDYEF-UHFFFAOYSA-N 0.000 description 1
- 241001068295 Replication defective viruses Species 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 210000000662 T-lymphocyte subset Anatomy 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- MKUXAQIIEYXACX-UHFFFAOYSA-N aciclovir Chemical compound N1C(N)=NC(=O)C2=C1N(COCCO)C=N2 MKUXAQIIEYXACX-UHFFFAOYSA-N 0.000 description 1
- 229960004150 aciclovir Drugs 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000003190 augmentative effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 102000005936 beta-Galactosidase Human genes 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 238000009566 cancer vaccine Methods 0.000 description 1
- 229940022399 cancer vaccine Drugs 0.000 description 1
- 230000034303 cell budding Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000012761 co-transfection Methods 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 102000003675 cytokine receptors Human genes 0.000 description 1
- 108010057085 cytokine receptors Proteins 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000008260 defense mechanism Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 108010078428 env Gene Products Proteins 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 229960005102 foscarnet Drugs 0.000 description 1
- 210000001652 frontal lobe Anatomy 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000012642 immune effector Substances 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 description 1
- 108010052968 leupeptin Proteins 0.000 description 1
- 238000000464 low-speed centrifugation Methods 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 201000006512 mast cell neoplasm Diseases 0.000 description 1
- 208000006971 mastocytoma Diseases 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- OHDXDNUPVVYWOV-UHFFFAOYSA-N n-methyl-1-(2-naphthalen-1-ylsulfanylphenyl)methanamine Chemical compound CNCC1=CC=CC=C1SC1=CC=CC2=CC=CC=C12 OHDXDNUPVVYWOV-UHFFFAOYSA-N 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 229950000964 pepstatin Drugs 0.000 description 1
- 108010091212 pepstatin Proteins 0.000 description 1
- FAXGPCHRFPCXOO-LXTPJMTPSA-N pepstatin A Chemical compound OC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)NC(=O)CC(C)C FAXGPCHRFPCXOO-LXTPJMTPSA-N 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000007505 plaque formation Effects 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 230000003393 splenic effect Effects 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000007910 systemic administration Methods 0.000 description 1
- 238000012385 systemic delivery Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 230000037455 tumor specific immune response Effects 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/245—Herpetoviridae, e.g. herpes simplex virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/76—Viruses; Subviral particles; Bacteriophages
- A61K35/763—Herpes virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
- A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/193—Colony stimulating factors [CSF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/20—Interleukins [IL]
- A61K38/208—IL-12
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0331—Animal model for proliferative diseases
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
- C12N2710/16632—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
- C12N2710/16641—Use of virus, viral particle or viral elements as a vector
- C12N2710/16643—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/108—Plasmid DNA episomal vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2840/00—Vectors comprising a special translation-regulating system
- C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
- C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
Definitions
- Induction of tumor-specific immunity is an attractive approach for cancer therapy because of the prospect of harnessing the body's own defense mechanisms, rather than using standard toxic therapeutic agents, to provide long-term protection against tumor existence, growth and recurrence. This strategy is attractive for its potential to destroy small metastatic tumors which may escape detection, and to provide immunity against recurrent tumors.
- an immunotherapy would depend on the presence of tumor-specific antigens and on the ability to induce a cytotoxic immune response that recognizes tumor cells which present antigens.
- Cytotoxic T lymphocytes CTL
- MHC major histocompatibility complex
- Roth et al. Adv. Immunol. 57: 281-351 (1994); Boon et al., Annu. Rev. Immunol. 12: 337-65 (1994).
- Some cancer vaccination strategies have focused on the use of killed tumor cells or lysates delivered in combination with adjuvants or cytokines. More recently, gene transfer of cytokines, MHC molecules, co-stimulatory molecules, or tumor antigens to tumor cells has been used to enhance the tumor cell's visibility to immune effector cells. Dranoff & Mulligan, Adv. Immunol. 58: 417-54 (1995).
- cancer vaccines have presented major difficulties, however.
- conventional approaches require obtaining and culturing a patient's autologous tumor cells for manipulation in vitro, irradiation and subsequent vaccination, or the identification and purification of a specific tumor antigen.
- the present invention provides a method of eliciting a systemic antitumor immune response in a patient who presents with or who is at risk of developing multiple metastatic tumors of a given cell type.
- the method comprises inoculating a tumor in the patient with a pharmaceutical composition consisting essentially of:
- HSV herpes simplex virus
- the virus replicates in dividing cells and exhibits attenuated replication in non-dividing cells.
- the virus is replication-defective.
- the virus is conditionally replication-competent.
- the virus is of a vaccine strain.
- the genome of the virus comprises at least one expressible nucleotide sequence coding for at least one immune modulator.
- the method comprises inoculating a tumor in the patient with a pharmaceutical composition comprising:
- (C) a pharmaceutically acceptable vehicle for the virus and defective vector, such that an immune response is induced that is specific for the tumor cell type and that kills cells of the inoculated tumor and of a non-inoculated tumor.
- the method comprises inoculating a tumor in the patient with a pharmaceutical composition comprising:
- HSV herpes simplex virus
- HSV herpes simplex virus
- the method comprises inoculating a tumor in the patient with a pharmaceutical composition comprising:
- a first herpes simplex virus that infects tumor cells but that does not spread in normal cells, wherein the genome of the first herpes simplex virus comprises at least one expressible nucleotide sequence coding for at least one immune modulator,
- a second herpes simplex virus that infects tumor cells but that does not spread in normal cells, wherein the genome of the second herpes simplex virus comprises at least one expressible nucleotide sequence coding for at least one immune modulator, and
- the method comprises inoculating a tumor in the patient with a pharmaceutical composition comprising:
- HSV herpes simplex virus
- the viral vector may be, for example, an adenoviral vector, a adenovirus-associated vector, a retroviral vector, or a vaccinia virus vector.
- Mutated viruses useful in the methods of the invention also are provided.
- a herpes simplex virus that is incapable of expressing both (i) a functional ⁇ 34.5 gene product and (ii) a ribonucleotide reductase, wherein the genome of the virus comprises at least one expressible nucleotide sequence encoding at least one immune modulator.
- a herpes simplex virus ICP4 mutant tsK the genome of which has been altered to incorporate at least one expressible nucleotide sequence coding for at least one immune modulator.
- compositions for effecting the methods of the present invention also are provided.
- a composition for eliciting a systemic antitumor immune response in a patient who presents with or who is at risk of developing multiple metastatic tumors of a given cell type comprises:
- composition for eliciting a systemic antitumor immune response in a patient who presents with or who is at risk of developing multiple metastatic tumors of a given cell type comprises:
- composition for eliciting a systemic antitumor immune response in a patient who presents with or who is at risk of developing multiple metastatic tumors of a given cell type comprises:
- FIG. 1C shows that increasing the intratumoral dose of G207 results in decreased bilateral tumor growth of CT26 tumors in BALB/c mice.
- the bars show the average of 6 animals per group.
- FIG. 5A shows plasmid pHCL-tk.
- FIG. 5B shows plasmid pHCIL12-tk.
- FIG. 6 shows the secretion of IL-12 in cells inoculated with dvIL12/G207.
- FIG. 8 shows the survival rate of mice post-inoculation with dvlacZ/G207, dvIL12/G207 or mock.
- FIG. 10 shows the survival rate of mice post-inoculation with dvlacZ/tsK, dvIL12/tsK or mock.
- the present invention provides a method of eliciting a systemic antitumor immune response in a patient presenting with, or at risk of developing, multiple metastatic tumors by inoculating at least one tumor with a mutated herpes simplex virus (HSV).
- HSV herpes simplex virus
- the inoculation invokes a highly specific antitumor immune response which kills cells of the inoculated tumor, as well as cells of distant, established, non-inoculated tumors.
- the ability to treat patients presenting with multiple metastatic tumors represents a significant advantage over conventional approaches which focus on the treatment of a single tumor mass.
- the efficacy of conventional cytotoxic viral vector-based approaches depends on the viral infection of all tumor cells in the patient. It is extremely difficult to obtain broad or systemic distribution of viral vectors in vivo, however, and therefore difficult to infect all tumor cells of a localized solid tumor, and virtually impossible to infect all tumor cells in a patient presenting with multiple metastatic tumors.
- the method of the present invention which does not require the targeting of a viral vector to every tumor cell, therefore offers a distinct improvement over these methods.
- With recent improvements in cancer therapy of primary tumors many patients survive longer and are at risk of developing multiple metastatic tumors. Accordingly, the ability to treat these patients effectively represents a needed improvement in cancer therapy.
- the viruses used in accordance with the present invention are mutated herpes simplex viruses that infect tumor cells but do not spread efficiently to or replicate efficiently in normal cells or tissue, thereby causing no disease or pathology in and of itself.
- a virus that replicates in dividing cells and exhibits attenuated replication in non-dividing cells is useful in accordance with the present invention, as is a virus that is replication-defective.
- the virus may be of type 1 (HSV-1) or type 2 (HSV-2).
- HSV-1 mutants have been used for local cytotoxic tumor therapy to destroy tumor cells in situ, yet spare normal tissue.
- the virus replicates in dividing cells and exhibits attenuated replication in non-dividing cells.
- U.S. Pat. No. 5,585,096 describes a suitable virus, illustrated by strain G207, which is incapable of expressing both (i) a functional ⁇ 34.5 gene product and (ii) a ribonucleotide reductase.
- G207 replicates in dividing cells, effecting a lytic infection with consequent cell death, but is highly attenuated in non-dividing cells, thereby targeting viral spread to tumors.
- G207 is non-neuropathogenic, causing no detectable disease in mice and non-human primates. Mineta et al., Nature Medicine 1: 938-43 (1995).
- the virus is replication-defective.
- exemplary of such a virus is tsK, a temperature-sensitive herpes simplex virus mutant in ICP4. Davison et al., J. Gen. Virol. 65: 859-63 (1984).
- the ability of tsK to replicate is temperature-dependent, with 31.5° C. permissive for replication, and 39.5° C. non-permissive.
- tsK can replicate with varying ability between these temperatures. Because body temperature is about 39.5° C., tsK is expected to be replication-defective in vivo. This has been confirmed by in vivo experiments with tsK in rats.
- the virus is conditionally replication-competent.
- An example of such a virus is G92A, whose ability to replicate is cell-type dependent. G92A is described in more detail in U.S. application Ser. No. 08/486,147, filed Jun. 7, 1995, the contents of which are incorporated herein by reference.
- the immunological properties of the mutated herpes simplex virus consist essentially of inducing an immune response that is specific for the tumor cell type and that kills cells of the inoculated tumor and of a non-inoculated tumor.
- the phrase “consisting essentially of” excludes another feature that would affect significantly a material aspect of the invention.
- the genome of the mutated virus does not comprise an expressible immune modulator, such as IL-2.
- other embodiments of the invention encompass mutant viruses whose genomes do comprise an expressible immune modulator.
- compositions consisting essentially of the herpes simplex virus and a pharmaceutically acceptable carrier, that is administered to a patient who suffers from or who is at risk of developing multiple, metastatic tumors.
- the composition is administered directly to the tumors cells in situ.
- the phrase “consisting essentially of” excludes a step or other feature that would affect significantly a material aspect of the invention.
- a composition of this embodiment would include, for example, the prescribed herpes simplex virus with no other virus or defective virus vector; this, because an additional virus would substantially complicate the inventive protocol.
- the invention also encompasses the administration of this composition in combination with another therapy, such as chemotherapy or radiation treatment.
- more than one mutated herpes simplex virus is administered.
- This embodiment can be effected by administering a single composition comprising more than one mutated herpes simplex virus and a pharmaceutically acceptable vehicle for the viruses, or by, administering more than one composition, each composition comprising at least one mutated herpes simplex virus and a pharmaceutically acceptable vehicle for the virus or viruses.
- a composition is administered that comprises (A) a first mutated herpes simplex virus, (B) a second mutated herpes simplex virus and (C) a pharmaceutically acceptable carrier for the viruses.
- a composition is administered that consists essentially of (A) a first mutated herpes simplex virus, (B) a second mutated herpes simplex virus and (C) a pharmaceutically acceptable carrier for the viruses.
- A a first mutated herpes simplex virus
- B a second mutated herpes simplex virus
- C a pharmaceutically acceptable carrier for the viruses.
- the phrase “consisting essentially of” excludes a step or other feature that would affect significantly a material aspect of the invention.
- this embodiment would entail, for example, the administration of the prescribed first and second herpes simplex viruses with no other virus or defective virus vector.
- the inoculation of a tumor with one or more mutated herpes simplex viruses in accordance with the present invention induces a systemic tumor-specific immune response that is specific for the cell type of the inoculated tumor and that kills cells of the inoculated tumor and of other, non-inoculated tumors.
- the induced cell death is observed, for example, as inhibited tumor growth or as reduced tumor size. In the examples set forth below, the induced cell death is observed as an inhibition of the growth of the inoculated tumor and of distant, established, non-inoculated tumors. In some instances, the tumors shrink to undetectable sizes.
- the immune response is correlated with cytotoxic T lymphocytes (CD8 + ) that recognize a major histocompatibility complex (MHC) class I-restricted peptide that is a dominant tumor antigen.
- CD8 + cytotoxic T lymphocytes
- MHC major histocompatibility complex
- the composition is administered directly to tumor cells of the patient, in situ. This can be accomplished by procedures known in the art, for example, by intratumoral inoculation during surgery, such as surgery for debulking a tumor, into external melanomas, or stereotactically into the tumor bed. Other approaches for targeting tumors also are appropriate. Generally, the maximum safe dose is administered at weekly intervals if the tumor is readily accessible, or is administered during surgery or tumor biopsy.
- the pharmaceutically acceptable vehicle for the virus can be selected from known pharmaceutically acceptable vehicles, and should be one in which the virus is stable.
- it can be a diluent, solvent, buffer, and/or preservative.
- An example of a pharmaceutically acceptable vehicle is phosphate buffer containing NaCl.
- Other pharmaceutically acceptable vehicles aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 15th Ed. Easton: Mack Publishing Co. pp 1405-1412 and 1461-1487 (1975) and THE NATIONAL FORMULARY XIV., 14th Ed. Washington: American Pharmaceutical Association (1975), the contents of which are hereby incorporated by reference.
- the lytic destruction or virally-induced death of tumor cells might release tumor antigens which then are picked up by APCs and carried to the draining lymph nodes. There they would be processed and presented to CD8 + T cells. Associative recognition of HSV-specific and tumor-specific antigens might also play a role in the strength of the response.
- Tumor cells infected with replication-competent HSV would have maturing virions budding from their cell membranes and may also process viral antigens for MHC class-I presentation likes APCs do.
- the HSV-infected tumor cells therefore might induce T cell-Mediated immune reactions directly. Some of the immune response induced by co-presentation of viral and tumor antigens may be triggered thereafter by only one of the co-expressed antigens.
- one or more immune modulators are delivered to the tumor cells in addition to the mutated herpes simplex virus described above.
- immune modulators useful in the present invention include cytokines, co-stimulatory molecules, and chemokines. Delivery of one or more immune modulators can be effected, for example, by means of a mutated herpes simplex virus that comprises one or more expressible nucleotide sequences encoding one or more cytokines or other immune-modulatory genes, or by means of more than one mutated herpes simplex virus, each of which comprises one or more expressible nucleotide sequences encoding one or more cytokines or other immune-modulatory genes.
- Non-herpes simplex virus vectors also can be used to effect delivery of one or more immune modulators.
- one or more adenoviral vectors, adenovirus-associated vectors, retroviral vectors, or vaccinia virus vectors comprising one or more expressible nucleotide sequences encoding one or more immune-modulatory genes can be used in accordance with this embodiment. See, e.g., Shawler et al., Adv. Pharacol. 40: 309-37 (1997), discussing gene transfer of immunostimulatory cytokines.
- the present invention also comprehends a situation where the patient receives both a mutated herpes simplex virus and a defective herpes simplex virus vector which contains the genes for one or more immune modulators, and where the former virus acts as a helper virus for the defective vector. Additionally, the invention encompasses the administration of one or more mutated herpes simplex viruses and more than one defective herpes simplex virus vectors, where each defective vector contains the genes for one or more immune modulators, and where the former virus or viruses act as helpers for the defective vectors.
- the immunological properties of the helper viruses consist essentially of inducing an immune response that is specific for the tumor cell type and that kills cells of the inoculated tumor and of a non-inoculated tumor.
- “consisting essentially of” excludes another feature that would affect significantly a material aspect of the invention. Accordingly, the use of this phrase excludes, for example, the administration of a helper virus vector that is capable of expressing an immune modulator, such as IL-2.
- immune modulators examples include IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-12, G-CSF, GM-CSF, IFN- ⁇ , IFN- ⁇ , TNF- ⁇ and B7. See, e.g., Purani et al., Adv. Pharmacol. 40: 259-89 (1997); Shawler et al., Adv. Pharmacol. 40: 309 (1997).
- IL-12 is exemplified in the discussion which follows. It is to be understood, however, that other immune modulators can be used in its place or in addition thereto.
- an immune modulator it is to be understood that the invention encompasses one or more immune modulators.
- the cytokine IL-12 is a heterodimeric cytokine, composed of 35 kD (p35) and 40 kD (p40) subunits, that binds to receptors present on NK and T cells.
- the high-affinity receptor is composed of two ⁇ -type cytokine receptor subunits that individually behave as low affinity receptors.
- IL-12 plays a multi-functional role in the immune system, augmenting the proliferation and cytotoxic activity of T cells and NK cells, regulating IFN- ⁇ production and promoting the development of CD4 + T helper (Th1) cells.
- IL-12 The antitumor activity of IL-12 has been demonstrated in a number of different murine tumor models, both solid and metastatic, with systemic administration of recombinant IL-12, fibroblasts or tumor cells engineered to secrete IL-12, and viral vectors expressing IL-12.
- IL-12 immunotherapy is less effective with other tumor cell lines such as CT26, C26, MCH-1-A1, and TS/A.
- Systemic delivery of rIL-12 has been shown to have potent antitumor effects in various animal models. Prolonged exposure to IL-12 can have deleterious side effects like those observed with many cytokines, however.
- tumors are modified in situ to make tumor cells a source of immune modulator production.
- Defective herpes simplex virus vectors are plasmid-based vectors which are unable to replicate on their own because they lack viral genes, but which contain specific HSV sequences that, in the presence of helper herpes simplex virus, support DNA replication and subsequent packaging into virus particles. Lim et al., BioTechniques 20(3): 460 (1996); Spaete and Frenkel, Cell 30: 295-304 (1982).
- the defective herpes simplex virus vector contains one or more nucleotide sequences encoding one or more cytokines or other immune modulators.
- any herpes simplex virus described above can be used as helper virus, such as a replication-competent virus, a replication-defective virus, or a conditionally replication-competent virus.
- each defective vector can contain multiple copies of the immune modulator gene.
- a defective vector containing an IL-12 gene can contain approximately 15 copies of the IL-12 gene (based on the size of the IL-12-containing plasmid), which can transduce both dividing and non-dividing cells at high efficiency.
- the viral DNA does not integrate into the infected cell genome, and with the CMV promoter driving IL-12 expression, expression is strong but transient.
- a defective is a defective.
- HSV vector is used to deliver one or more immune modulators such as IL-12 in combination with G207 as a helper virus.
- a defective HSV vector is used to deliver one or more immune modulators such as IL-12 in combination with tsK as a helper virus.
- the construction of defective herpes virus vectors and their use with helper viruses is known in the art. For example, see Spaete & Frankel, supra, and Geller et al., Proc. Nat'l Acad. Sci. USA 87: 8950-54 (1990).
- the defective IL-12-containing vector infects a number of different tumor cells which then produce and secrete IL-12 in vivo.
- the IL-12 acts as an adjuvant for the immune response elicited by the herpes simplex virus.
- the enhanced immune response is correlated with heightened induction of tumor-specific CTL activity and IFN- ⁇ production by splenocytes, as described in more detail in the examples below.
- one or more defective herpes simplex virus vectors containing one or more immune modulators and one or more helper herpes simplex viruses kills cells of the inoculated tumor and of other, non-inoculated tumors. This antitumor effect is significantly greater than that observed when a tumor is inoculated with a mutated herpes simplex virus alone, revealing a synergistic effect.
- the immune response elicited in accordance with the present invention kills cells of the inoculated tumor and also kills non-inoculated tumor cells, including cells of distant, non-inoculated tumors.
- This effect makes this method particularly useful for treating patients presenting with multiple metastatic tumors of a given cell type. It also represents an improvement in the treatment of localized, non-metastatic tumors because the method kills tumor cells that are not directly targeted by the administered virus.
- tumor can be treated in accordance with the present invention, including non-metastatic tumors, tumors with metastatic potential, and tumors already demonstrating an ability to metastasize.
- tumor cell types include astrocytoma, oligodendroglioma, meningioma, neurofibroma, glioblastoma, ependymoma, Schwannoma, neurofibrosarcoma, and medulloblastoma cell types.
- the invention also is useful in treating melanoma cells, pancreatic cancer cells, prostate carcinoma cells, head and neck cancer cells, breast cancer cells, lung cancer cells, colon cancer cells, lymphoma cells, hepatoma cells, ovarian cancer cells, renal cancer cells, neuroblastomas, squamous cell carcinomas, sarcomas, and mesothelioma and epidermoid carcinoma cells.
- the antitumor efficacy of G207 was evaluated in a bilateral, established subcutaneous tumor model with CT26 cells as described below.
- CT26 The murine colorectal carcinoma CT26 cell line has been widely used as a syngeneic tumor model to study immunotherapy.
- CT26 is a transplantable colon epithelial tumor induced by intrarectal injections of N-nitroso-N-methylurethane in female BALB/c mice (H-2 d ). Corbett et al., Cancer Res. 35: 2434-39 (1975).
- CT26 is poorly immunogenic: 10 3 -10 4 cells can cause a lethal tumor and do not induce detectable tumor-specific CTL.
- AH1 a nonmutated nonamer derived from the envelop protein (gp70) of an endogenous ecotropic murine leukemia provirus (MuLV), env-1, has been identified as the immunodominant MHC class I-restricted antigen for CT26.
- MoLV ecotropic murine leukemia provirus
- Adoptive transfer of peptide-specific CTL lines has been able to cure established subcutaneous CT26 tumors, demonstrating the correlation between induction of tumor-specific CTL and an antitumor effect.
- Herpes simplex virus does not grow in many rat cells, and attenuated viruses like G207 do not grow well in many mouse tumors either. This is in contrast to their excellent growth in most human tumor lines.
- studies in human tumor lines require the use of athymic mice.
- CT26 was chosen as a model cell line after several years of trying to find a good syngeneic system for studying the immune effects of attenuated conditionally replicated herpes vectors, such as G207.
- mice were injected subcutaneously in the bilateral flanks of female BALB/c mice (National Cancer Institute (Rockville, Md.)). When subcutaneous tumors were palpably growing (approximately 5 mm in diameter), mice were unilaterally inoculated into the right side tumor with either G207 virus in 50 ⁇ l of virus buffer (150 mM NaCl, 20 mM Tris, pH 7.5) and modified Eagle's medium (MEM) (1:1), or with 50 ⁇ l of mock-infected extract (“mock”), prepared from mock-infected cells using the same procedures as those used for the virus inoculum. A second injection of the same composition was given 7 days later in some experiments. Tumor size was measured by external caliper. All animal procedures were approved by the Georgetown University Animal Care and Use Committee.
- inoculation with G207 resulted in a reduction in tumor growth of both the inoculated tumors (Rt), as well as of their non-inoculated contralateral counterparts (Lt) when compared to mock-inoculated controls (p ⁇ 0.0005 (Rt) and p ⁇ 0.001 (Lt) on day 21 postinfection; unpaired t-test).
- Rt inoculated tumors
- Lt non-inoculated contralateral counterparts
- lacZ expression from G207 was detected by X-gal histochemistry in the inoculated tumor but not the non-inoculated tumor.
- FIG. 1A A single unilateral intratumoral inoculation with 5 ⁇ 10 7 pfu of G207 caused a large reduction in bilateral tumor growth ( FIG. 1A ), comparable to the double inoculation with 7 ⁇ 10 5 pfu ( FIG. 1C ).
- effector cells were generated in vitro from splenocytes obtained 12 days after the first virus (G207) inoculation and tested in a 51 Cr release assay.
- A20 is a B cell lymphoma cell line (Ig + , Ia + , H-2 d ) derived from a spontaneous reticulum cell neoplasm in BALB/c mice. Kim et al., J. Immunol. 122: 549-54 (1979). It is capable of presenting protein antigen to MHC-restricted antigen-reactive T lymphocytes. Glimcher, et al., J. Exp. Med. 155: 445-59 (1982).
- mice treated intratumorally with G207 generated a highly specific CTL response against CT26 cells but not against A20 lymphoma cells (also H-2 d ). No specific CTL response was detected in mice treated intradermally with G207 or intratumorally with mock extract. There was a small non specific CTL response (against A20 and CT26) induced in mock-inoculated mice.
- AH1 the nonamer SPSYVYHQF, is the immunodominant peptide from CT26, presented by the MHC class I L d molecule.
- the L d -binding AH1 peptide is derived from gp70, one of two env gene products of the endogenous MuLV.
- AH1 peptide was synthesized by Peptide Technologies (Washington, D.C.) to a purity of >99% as determined by HPLC and amino acid analysis.
- H-2L d -restricted P815AB.35-43 is the immunodominant peptide derived from murine mastocytoma P815 cells. Van den Eynde et al., J. Exp. Med. 173: 1373-84 (1991).
- Effector cells from intratumoral G207-inoculated mice exhibited specific lysis of CT26 cells and of A20 cells pulsed with L d -restricted peptide AH1, but not of A20 cells pulsed with L d -restricted peptide P815AB.
- the in vitro CTL activity was completely abrogated by depletion of CD8 + cells, but not by depletion of CD4 + cells.
- Intradermal inoculation with G207 virus or intratumoral inoculation with mock extract did not enhance the activation of specific T cells against CT26 tumors.
- in vivo priming against tumors that express endogenous antigens by intratumoral inoculation of G207 induced an antigenic peptide-specific CTL response.
- M3 mouse melanoma cells (3 ⁇ 10 5 ) were inoculated bilaterally into the flanks of DBA/2 mice. When the tumors were 5 mm in maximal diameter, the right flank tumor was inoculated one time with either 5 ⁇ 10 7 pfu of G207 or an equivalent amount of mock Vero cell preparation (as a negative control).
- Inoculation with G207 inhibited the growth of the inoculated tumor (p ⁇ 0.0005), and also significantly inhibited the growth of the non-inoculated tumor (p ⁇ 0.02).
- FIG. 2 Inoculation with G207 inhibited the growth of the inoculated tumor (p ⁇ 0.0005), and also significantly inhibited the growth of the non-inoculated tumor (p ⁇ 0.02).
- Mouse N18 neuroblastoma cells were subcutaneously implanted bilaterally into syngeneic A/J mice. Eight days after tumor implantation, 10 7 pfu of G207 or mock were injected into the left tumor. In six of eight animals, inoculation with G207 resulted in the disappearance of the tumors on both sides.
- FIG. 3 Mouse N18 neuroblastoma cells were subcutaneously implanted bilaterally into syngeneic A/J mice. Eight days after tumor implantation, 10 7 pfu of G207 or mock were injected into the left tumor. In six of eight animals, inoculation with G207 resulted in the disappearance of the tumors on both sides. FIG. 3 .
- N18 neuroblastoma cells were subcutaneously implanted bilaterally into the left flank of A/J mice. Three days later, N18 neuroblastoma cells were intracerebrally implanted into the right frontal lobe of the mice. On days 10 and 13, the subcutaneous tumors only were injected with G207 (11 mice) or mock (11 mice). Within 35 days of cerebral implantation, all mock-treated mice died from or had intracerebral tumors. Four out of eleven mice treated with G207 had no intracerebral tumors, and one G207-treated mouse was a long-term survivor. G207 treatment inhibited growth of distant, intracerebral tumors and increased the survival of tumor-bearing animals (P ⁇ 0.05 by Wilcox test).
- N18 cells Ten A/J mice with no previous exposure to N18 cells (naive group), thirty A/J mice that had spontaneously rejected prior subcutaneous injections of N18 cells (rejection group) and twelve A/J mice that previously had established N18 subcutaneous tumors that were cured by intratumoral injection of G207 (cured group) were subcutaneously injected with N18 cells. None of the animals of the cured group showed any sign of tumor growth, whereas a large number of animals of the naive and rejection groups showed significant tumor growth.
- Mouse CT26 colon carcinoma cells were subcutaneously implanted bilaterally into syngeneic BALB/c mice. 10 5 pfu of tsK, a temperature-sensitive herpes simplex virus mutant in ICP4, or mock was injected into the right tumor, and a second inoculation of the same composition was given seven days later (day 7). Inoculation with tsK resulted in significant inhibition of tumor growth in both tumors (p ⁇ 0.05 on day 21).
- FIG. 4 .
- the murine colorectal carcinoma cell line CT26 was used to evaluate the antitumor efficacy of a defective vector containing IL-12 and G207 as the helper virus.
- pHCIL12-tk and pHCL-tk Two amplicon plasmids of similar size were constructed, pHCIL12-tk and pHCL-tk, which encoded the two subunits of murine IL-12 (p40 and p35) or lacZ, respectively, under control of the CMV IE promoter (see FIGS. 5A and 5B ). Since IL-12 is functional as a heterodimer, both subunits were expressed from a single defective vector, as a bicistronic message, by means of an internal ribosome entry site (IRES).
- IFS internal ribosome entry site
- the double-cassette amplicon plasmid pHCL-tk was constructed by inserting the HSV-1 thymidine kinase (TK) gene and the blunt-ended BamH1 fragment from pHSV-106 (Life Technologies, Inc., Rockville, Md.) into the blunt-ended Spe I site of pHCL ( FIG. 5A ).
- TK HSV-1 thymidine kinase
- IL-12 p35 and an IRES from equine encephalomyocarditis virus (EMCV) from DFG-mIL-12 (IRES-p35), and BamH1 fragment from DFG-mIL12 were subcloned into LITMUS 28 (New England Biolabs, MA) at the BglII/BamH1 site to generate p40-IRES-35.
- the IL-12 encoding double-cassette amplicon plasmid pHCIL12-tk was constructed by insertion of the p40-IRES-p35 cassette, SnaB1/AflII fragment, into the blunt-ended SalI site of pSR-ori and then inserting the HSV TK blunt-ended BamH1 fragment into the blunt-ended SphI site to produce pHCIL12-tk.
- FIG. 5B The IL-12 encoding double-cassette amplicon plasmid pHCIL12-tk was constructed by insertion of the p40-IRES-p35 cassette, SnaB1/AflII fragment, into the blunt-ended SalI site of pSR-ori and then inserting the HSV TK blunt-ended BamH1 fragment into the blunt-ended SphI site to produce pHCIL12-tk.
- FIG. 5B The IL-12 encoding double-cassette amplicon plasmid pHCIL12-tk was constructed by
- G207 containing deletions in both copies of the ⁇ 34.5 gene and an E. coli lac Z insertion inactivating the ICP6 gene, was used as helper virus for the generation of defective vector (dv) stocks.
- Vero cells were co-transfected with purified amplicon plasmid DNA (pHCIL12-tk and pHCL-tk) and G207 viral DNA using lipofectAMINETM (Life Technologies, Inc., Rockville, Md.), as described by the manufacturer, and then cultured at 34.5° C. until they exhibited complete cytopathic effect. Virus was then harvested and passaged at a 1:4 dilution in Vero cells until inhibition of helper virus replication was observed.
- the IL-12 containing defective vector is called dvIL12/G207 and the lacZ containing defective vector is called dvlacZ/G207.
- G207 helper virus titer was expressed as the number of pfus after plaque assay on Vero cells at 34.5° C.
- IL-12 expression was determined and the passage with highest level was used (passage 4) with a G207 helper virus titer of 5 ⁇ 10 7 pfu/ml.
- the titer of dvlacZ/G207 determined by counting X-gal (5-bromo-4-chloro-3-indolyl- ⁇ -D-glactopyranoside) histochemistry positive single cells (defective particle units, dpu) after formation of plaques by G207, was 5 ⁇ 10 6 dpu/ml and 5 ⁇ 10 7 pfu/ml of helper virus.
- African green monkey kidney (Vero) cells were cultured in DMEM containing 10% calf serum (CS).
- MC-38 mouse colon adenocarcinoma, Harding-Passey mouse melanoma, MDA-MB-435 human breast adenocarcinoma, and CT26 cells were grown in DMEM containing 10% heat-inactivated FCS (Hyclone, Logan, Utah) and penicillin-streptomycin (Sigma Chemical Co, St Louis, Mo.).
- A20 a B cell lymphoma cell line (Ig + , Ia + , H-2 d ) derived from a spontaneous reticulum cell neoplasm in BALB/c mice (American Type Culture Collection, Rockville, Md., ATCC TIB 208) was grown in RPMI 1640 containing 10% heat-inactivated FCS, 50 ⁇ M of 2-ME, 2 mM glutamine, 20 mM Hepes buffer, and penicillin-streptomycin.
- the expression and secretion of IL-12 was determined by ELISA assay after infection of tumor cells in culture at a multiplicity of infection (MOI) of 1 pfu per cell.
- 96-well plates coated with an anti-mouse IL-12 mAb (9A5) were incubated overnight at room temperature with the test samples. After washes, the plates were incubated with peroxidase-labelled anti-mouse IL-12 p40 Ab (5C3) for 2 hours and then were developed. Absorbance was measured at 450 nm.
- FIG. 6 No IL-12 was detected in the supernatants of uninfected tumor cell cultures or those infected with dvlacZ/G207. Levels of IL-12 synthesis and secretion peaked 1 day after dvIL12/G207 infection of CT26 cells and decreased to undetectable levels by 3 days post-infection, likely due to cell death.
- BALB/c and BALB/c (nu/nu) mice were obtained from the National Cancer Institute or Charles River (Wilmington, Mass.). All animal procedures were approved by the Georgetown University Animal Care and Use Committee.
- CT26 tumor cells (1 ⁇ 10 5 ) were injected subcutaneously (s.c.) in the bilateral flanks of mice.
- s.c. tumors were palpably growing (approximately 5 mm in maximal diameter)
- mice were unilaterally inoculated into the right side tumor with either 50 ⁇ l of defective HSV vector (7 ⁇ 10 5 pfu of helper virus) in virus buffer (150 mM NaCl, 20 mM Tris, pH 7.5) or 50 ⁇ l virus buffer, followed by a second injection of the same composition 7 days later. Where indicated, mock extract was used in place of virus buffer.
- DvlacZ/G207 rather than helper virus G207 alone was used as a control for dvIL12/G207 inoculation so that differences in viral factors (i.e., particle:pfu ratio) present in defective vector stocks versus G207 stocks would be accounted for.
- FIG. 7 Two out of six of the dvIL12/G207 inoculated tumors regressed to an undetectable size. Inoculation with dvlacZ/G207 also resulted in a significant reduction in tumor growth of both inoculated and non-inoculated tumors compared to controls, although to a much lesser extent than dvIL12/G207.
- FIG. 7 Two out of six of the dvIL12/G207 inoculated tumors regressed to an undetectable size. Inoculation with dvlacZ/G207 also resulted in a significant reduction in tumor growth of both inoculated and non-inoculated tumors compared to controls, although to a much lesser extent than dvIL12/G207.
- mice also were followed for survival, where sacrifice occurred when either of the bilateral tumors became larger than 18 mm in diameter. Survival of the defective vector-treated animals is therefore reflective of the growth of the non-inoculated tumors and was significantly longer than control animals. Mice treated unilaterally with dvIL12/G207 survived longer than dvlacZ/G207 treated mice ( FIG. 8 ). IL-12 was detected in the dvIL12/G207 inoculated tumors one and five days post-inoculation (approximately 50-100 pg/tumor), with no IL-12 detected in the serum.
- CT26 tumors grew somewhat more rapidly in the athymic mice than in the immune-competent mice.
- mice were inoculated with dvIL12/G207 or dvlacZ/G207 intratumorally when s.c. tumors reached approximately 5 mm in maximal diameter, and a second inoculation of the same composition was given seven days later.
- Single-cell suspensions of splenocytes were cultured in RPMI 1640 medium with 10% inactivated FCS, 50 ⁇ M 2-ME, 2 mM glutamine, 20 mM Hepes, and penicillin-streptomycin in 24-well plates at a concentration of 3 ⁇ 10 6 cells/ml.
- either 1 ⁇ 10 6 inactivated CT26 cells or 1 ⁇ g/ml of peptide AH1 was added to the medium.
- CT26 tumor cells were incubated for 1 hour in culture medium containing 100 ⁇ g/ml of mitomycin C and then washed 2 times. Effector cells were harvested after 6 days of in vitro culture.
- 51 Cr release assays were performed as described above. In brief, target cells were incubated with 50 ⁇ Ci of Na 51 CrO 4 ( 51 Cr) for 60 min. A20 cells were pulsed with 1 ⁇ g/ml of the L d -restricted peptides AH1 or P815AB for 1 h before labeling. Target cells were then mixed with effector cells for 4 h at the E/T ratios indicated. The amount of 51 Cr release was determined by ⁇ counting, and the percent specific lysis was calculated from triplicate samples as follows:
- Splenocytes were isolated five days after the second inoculation of dvIL12/G207 or dvlacZ/G207 and tested for IFN- ⁇ production by ELISA and splenic T lymphocyte subsets by FACS analysis. Briefly, single-cell suspensions of splenocytes were washed and resuspended in RPMI 1640 medium containing 10% inactivated FCS. Cells (3 ⁇ 10 6 /ml) were cultured in 24-well plates for 24 h. Supernatants were collected and assayed by a sandwich ELISA using anti-IFN- ⁇ Ab pairs obtained from Endogen (Woburn, Mass.).
- Defective vectors containing IL-12 and tsK or lacZ and tsK were prepared.
- Defective vector plasmids pHCIL12-tk and pHCL-tk were prepared as described above.
- Defective vectors were generated by co-transfection of Vero cells with helper virus tsK DNA and pHCIL12-tk or pHCL-tk. Transfected cells were incubated at 31.5° C. (a replication-permissive temperature for tsK) until total cytopathic effect was observed. The cells then were passaged as described above for G207 helper virus. See also Kaplitt et al., Mol. Cell. Neurosci. 2: 320-30 (1991).
- the defective vector containing IL-12 is called dvIL12/tsK and the defective vector containing lacZ is called dvlacZ/tsK.
- CT26 mouse colon carcinoma cells were subcutaneously implanted bilaterally into syngeneic BALB/c mice, as described above.
- the right tumor was inoculated with either dvlacZ/tsK, dvIL12/tsK or mock, and a second inoculation of the same composition was given seven days later.
- Inoculation with dvlacZ/tsK resulted in a significant inhibition of tumor growth in both tumors (p ⁇ 0.01 on day 22).
- Inoculation with dvIL12/tsK resulted in greater inhibition of tumor growth in both tumors compared to dvlacZ/tsK-inoculated tumor (p ⁇ 0.001).
- FIG. 9 is .
- mice inoculated mice were sacrificed when they became moribund or when their tumors reached greater than 18 mm in diameter. As shown in FIG. 10 , mice inoculated with dvlacZ survived significantly longer than mice inoculated with mock (p ⁇ 0.01), and mice inoculated with dvIL12/tsK survived significantly longer than mice inoculated with dvlac/tsK or mock (p ⁇ 0.01).
- Harding-Passey melanoma cells were subcutaneously implanted into the bilateral flanks of C57BL/6 mice.
- the tumors were about 5 mm in maximal diameter (day 0)
- the right flank tumors were injected with defective vector dvlacZ/tsk (generated from the amplicon plasmid pHCL-tk and expressing E. coil lacZ) or dvGMCSF/tsK (generated from the amplicon plasmid pHCGMCSF-tk, whose structure is the same as pHCIL12-tk except it contains mouse GM-CSF cDNA in place of IL-12 DNA; expression of GM-CSF was detected by ELISA) and helper tsK virus, or with virus buffer.
- Mice treated with dvGMCSF/tsK showed increased survival over mice treated with dvlacZ/tsk or buffer, and showed decreased tumor growth in both bilateral tumors.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Zoology (AREA)
- Virology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Mycology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Oncology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
- Induction of tumor-specific immunity is an attractive approach for cancer therapy because of the prospect of harnessing the body's own defense mechanisms, rather than using standard toxic therapeutic agents, to provide long-term protection against tumor existence, growth and recurrence. This strategy is attractive for its potential to destroy small metastatic tumors which may escape detection, and to provide immunity against recurrent tumors.
- In principle, an immunotherapy would depend on the presence of tumor-specific antigens and on the ability to induce a cytotoxic immune response that recognizes tumor cells which present antigens. Cytotoxic T lymphocytes (CTL) recognize major histocompatibility complex (MHC) class I molecules complexed to peptides derived from cellular proteins presented on the cell surface, in combination with co-stimulatory molecules. Mueller et al., Annu. Rev. Immunol. 7: 445-80 (1989). In fact, tumor-specific antigens have been detected in a range of human tumors. Roth et al., Adv. Immunol. 57: 281-351 (1994); Boon et al., Annu. Rev. Immunol. 12: 337-65 (1994).
- Some cancer vaccination strategies have focused on the use of killed tumor cells or lysates delivered in combination with adjuvants or cytokines. More recently, gene transfer of cytokines, MHC molecules, co-stimulatory molecules, or tumor antigens to tumor cells has been used to enhance the tumor cell's visibility to immune effector cells. Dranoff & Mulligan, Adv. Immunol. 58: 417-54 (1995).
- The therapeutic use of “cancer vaccines” has presented major difficulties, however. In particular, conventional approaches require obtaining and culturing a patient's autologous tumor cells for manipulation in vitro, irradiation and subsequent vaccination, or the identification and purification of a specific tumor antigen.
- It is therefore an object of the present invention to provide a method of eliciting a systemic antitumor immune response in a patient who presents with or who is at risk of developing multiple metastatic tumors without manipulating the patient's autologous tumor cells or identifying or purifying specific antigens.
- It is also an object of the present invention to provide vectors for effecting this method.
- In accomplishing these and other objectives, the present invention provides a method of eliciting a systemic antitumor immune response in a patient who presents with or who is at risk of developing multiple metastatic tumors of a given cell type. In accordance with one aspect of the invention, the method comprises inoculating a tumor in the patient with a pharmaceutical composition consisting essentially of:
- (A) a herpes simplex virus (HSV) that infects tumor cells but that does not spread in normal cells, and
- (B) a pharmaceutically acceptable vehicle for the virus, such that an immune response is induced that is specific for the tumor cell type and that kills cells of the inoculated tumor and of a non-inoculated tumor. In accordance with one embodiment, the virus replicates in dividing cells and exhibits attenuated replication in non-dividing cells. In accordance with another embodiment, the virus is replication-defective. In accordance with yet another embodiment, the virus is conditionally replication-competent. In accordance with another embodiment, the virus is of a vaccine strain. In accordance with one embodiment, the genome of the virus comprises at least one expressible nucleotide sequence coding for at least one immune modulator.
- In accordance with another aspect of the invention, the method comprises inoculating a tumor in the patient with a pharmaceutical composition comprising:
- (A) a herpes simplex virus that infects tumor cells but that does not spread in normal cells, and whose immunological properties consist essentially of inducing an immune response that is specific for the tumor cell type and that kills cells of the inoculated tumor and of a non-inoculated tumor,
- (B) a defective herpes simplex virus vector containing at least one expressible nucleotide sequence encoding at least one immune modulator, and
- (C) a pharmaceutically acceptable vehicle for the virus and defective vector, such that an immune response is induced that is specific for the tumor cell type and that kills cells of the inoculated tumor and of a non-inoculated tumor.
- In accordance with another aspect of the invention, the method comprises inoculating a tumor in the patient with a pharmaceutical composition comprising:
- (A) a first herpes simplex virus (HSV) that infects tumor cells but that does not spread in normal cells, and whose immunological properties consist essentially of inducing an immune response that is specific for the tumor cell type and that kills cells of the inoculated tumor and of a non-inoculated tumor,
- (B) a second herpes simplex virus (HSV) that infects tumor cells but that does not spread in normal cells, and
- (C) a pharmaceutically acceptable vehicle for the viruses, such that an immune response is induced that is specific for the tumor cell type and that kills cells of the inoculated tumor and of a non-inoculated tumor.
- In accordance with another aspect of the present invention, the method comprises inoculating a tumor in the patient with a pharmaceutical composition comprising:
- (A) a first herpes simplex virus (HSV) that infects tumor cells but that does not spread in normal cells, wherein the genome of the first herpes simplex virus comprises at least one expressible nucleotide sequence coding for at least one immune modulator,
- (B) a second herpes simplex virus (HSV) that infects tumor cells but that does not spread in normal cells, wherein the genome of the second herpes simplex virus comprises at least one expressible nucleotide sequence coding for at least one immune modulator, and
- (C) a pharmaceutically acceptable vehicle for the viruses, such that an immune response is induced that is specific for the tumor cell type and that kills cells of the inoculated tumor and of a non-inoculated tumor.
- In accordance with another aspect of the present invention, the method comprises inoculating a tumor in the patient with a pharmaceutical composition comprising:
- (A) a herpes simplex virus (HSV) that infects tumor cells but that does not spread in normal cells,
- (B) a viral vector comprising at least one expressible nucleotide sequences coding for at least one immune modulator, and
- (C) a pharmaceutically acceptable vehicle for the virus and viral vector, such that an immune response is induced that is specific for the tumor cell type and that kills cells of the inoculated tumor and of a non-inoculated tumor. The viral vector may be, for example, an adenoviral vector, a adenovirus-associated vector, a retroviral vector, or a vaccinia virus vector.
- Mutated viruses useful in the methods of the invention also are provided. In accordance with one aspect of the invention, there is provided a herpes simplex virus that is incapable of expressing both (i) a functional γ34.5 gene product and (ii) a ribonucleotide reductase, wherein the genome of the virus comprises at least one expressible nucleotide sequence encoding at least one immune modulator. In accordance with another aspect of the invention, there is provided a herpes simplex virus ICP4 mutant tsK, the genome of which has been altered to incorporate at least one expressible nucleotide sequence coding for at least one immune modulator.
- Compositions for effecting the methods of the present invention also are provided. In accordance with one aspect of the invention, a composition for eliciting a systemic antitumor immune response in a patient who presents with or who is at risk of developing multiple metastatic tumors of a given cell type comprises:
- (A) a herpes simplex virus that is incapable of expressing both (i) a functional γ34.5 gene product and (ii) a ribonucleotide reductase, and
- (B) a defective herpes simplex virus vector containing at least one expressible nucleotide sequence encoding at least one immune modulator.
- In accordance with another aspect of the invention, a composition for eliciting a systemic antitumor immune response in a patient who presents with or who is at risk of developing multiple metastatic tumors of a given cell type comprises:
- (A) a herpes simplex virus that is replication-defective, and whose immunological properties consist essentially of inducing an immune response that is specific for the tumor cell type and that kills cells of the inoculated tumor and of a non-inoculated tumor, and
- (B) a defective herpes simplex virus vector containing at least one expressible nucleotide sequence encoding at least one immune modulator.
- In accordance with yet another aspect of the invention, a composition for eliciting a systemic antitumor immune response in a patient who presents with or who is at risk of developing multiple metastatic tumors of a given cell type comprises:
- (A) a herpes simplex virus that is conditionally replication-competent, and
- (B) a defective herpes simplex virus vector containing at least one expressible nucleotide sequence encoding at least one immune modulator.
- These and other objects and aspects of the invention will become apparent to the skilled artisan in view of the teachings contained herein.
-
FIG. 1A shows that intratumoral inoculation of CT26 tumors in BALB/c mice with G207 inhibits growth of the inoculated tumor (rt) and of a non-inoculated tumor at a distant site (it). Bars represent means±SEM of 6 mice per group. Tumor Volume=(width×length×height). -
FIG. 1B shows that intradermal inoculation of CT26 tumors in BALB/c mice with G207 has no significant effect on tumor growth. Bars represent means±SEM of 6 mice per group. Tumor Volume=(width×length×height). -
FIG. 1C shows that increasing the intratumoral dose of G207 results in decreased bilateral tumor growth of CT26 tumors in BALB/c mice. The bars show the average of 6 animals per group. -
FIG. 2 shows that intratumoral inoculation of M3 mouse melanoma cells in DBA/2 mice with G207 inhibits the growth of the inoculated tumor (rt) and a distant non-inoculated tumor (lt). Bars represent means±SEM of 6 or 7 mice per group. Tumor Volume=(width×length×height). -
FIG. 3 shows that intratumoral inoculation of mouse N18 neuroblastoma cells in syngeneic A/J mice with G207 inhibits the growth of the inoculated tumor (Left Tumor) and a distant non-inoculated tumor (Right Tumor). Bars represent means±SEM of 8 mice per group. Tumor Volume=(width×length×height). -
FIG. 4 shows that intratumoral inoculation of CT26 tumors in BALB/c mice with tsK inhibits the growth of the inoculated tumor (Rt) and a distant non-inoculated tumor (Lt). Bars represent means±SEM of 6 mice per group. Tumor Volume=(width×length×height). -
FIG. 5A shows plasmid pHCL-tk.FIG. 5B shows plasmid pHCIL12-tk. -
FIG. 6 shows the secretion of IL-12 in cells inoculated with dvIL12/G207. -
FIG. 7 shows that intratumoral inoculation of CT26 tumors in BALB/c mice with dvlacZ/G207 or dvIL12/G207 inhibits the growth of the inoculated tumor (Rt) and a distant non-inoculated tumor (Lt). Bars represent means±SEM of 6 mice per group. Tumor Volume=(width×length×height). -
FIG. 8 shows the survival rate of mice post-inoculation with dvlacZ/G207, dvIL12/G207 or mock. -
FIG. 9 shows that inoculation of CT26 tumors in BALB/c mice with dvIL12/tsK or dvlacZ/tsK inhibits the growth of the inoculated tumor (Rt) and a distant non-inoculated tumor (Lt). Bars represent means±SEM of 6 mice per group. Tumor Volume=(width×length×height). -
FIG. 10 shows the survival rate of mice post-inoculation with dvlacZ/tsK, dvIL12/tsK or mock. - A new and improved approach for eliciting a systemic immune response in patients presenting with multiple metastatic tumors has been developed. In accordance with these developments, the present invention provides a method of eliciting a systemic antitumor immune response in a patient presenting with, or at risk of developing, multiple metastatic tumors by inoculating at least one tumor with a mutated herpes simplex virus (HSV). The inoculation invokes a highly specific antitumor immune response which kills cells of the inoculated tumor, as well as cells of distant, established, non-inoculated tumors.
- The ability to treat patients presenting with multiple metastatic tumors represents a significant advantage over conventional approaches which focus on the treatment of a single tumor mass. The efficacy of conventional cytotoxic viral vector-based approaches depends on the viral infection of all tumor cells in the patient. It is extremely difficult to obtain broad or systemic distribution of viral vectors in vivo, however, and therefore difficult to infect all tumor cells of a localized solid tumor, and virtually impossible to infect all tumor cells in a patient presenting with multiple metastatic tumors. The method of the present invention, which does not require the targeting of a viral vector to every tumor cell, therefore offers a distinct improvement over these methods. Moreover, with recent improvements in cancer therapy of primary tumors, many patients survive longer and are at risk of developing multiple metastatic tumors. Accordingly, the ability to treat these patients effectively represents a needed improvement in cancer therapy.
- The viruses used in accordance with the present invention are mutated herpes simplex viruses that infect tumor cells but do not spread efficiently to or replicate efficiently in normal cells or tissue, thereby causing no disease or pathology in and of itself. For example, a virus that replicates in dividing cells and exhibits attenuated replication in non-dividing cells is useful in accordance with the present invention, as is a virus that is replication-defective. The virus may be of type 1 (HSV-1) or type 2 (HSV-2). Various HSV-1 mutants have been used for local cytotoxic tumor therapy to destroy tumor cells in situ, yet spare normal tissue. Mineta et al., Nature Medicine 1: 938-43 (1995); Martuza et al., Science 252: 854-56 (1991); Boviatsis et al., Gene Therapy 1: 323-331 (1994); Randazzo et al., Virology 211: 94-101 (1995); Andreansky et al., Proc. Natl. Acad. Sci. USA 93: 11313-18 (1996). Any of these mutants can be used in accordance with the present invention, as can vaccine strains of HSV. A number of anti-viral drugs (i.e., acyclovir and foscarnet) against herpes simplex virus are available that would allow unforeseen viral spread to be treated.
- In a preferred embodiment of the present invention, the virus replicates in dividing cells and exhibits attenuated replication in non-dividing cells. For instance, U.S. Pat. No. 5,585,096 describes a suitable virus, illustrated by strain G207, which is incapable of expressing both (i) a functional γ34.5 gene product and (ii) a ribonucleotide reductase. (The contents of U.S. Pat. No. 5,585,096 are incorporated herein by reference.) G207 replicates in dividing cells, effecting a lytic infection with consequent cell death, but is highly attenuated in non-dividing cells, thereby targeting viral spread to tumors. G207 is non-neuropathogenic, causing no detectable disease in mice and non-human primates. Mineta et al., Nature Medicine 1: 938-43 (1995).
- Pursuant to another aspect of the present invention, the virus is replication-defective. Exemplary of such a virus is tsK, a temperature-sensitive herpes simplex virus mutant in ICP4. Davison et al., J. Gen. Virol. 65: 859-63 (1984). The ability of tsK to replicate is temperature-dependent, with 31.5° C. permissive for replication, and 39.5° C. non-permissive. tsK can replicate with varying ability between these temperatures. Because body temperature is about 39.5° C., tsK is expected to be replication-defective in vivo. This has been confirmed by in vivo experiments with tsK in rats.
- In accordance with another aspect of the present invention, the virus is conditionally replication-competent. An example of such a virus is G92A, whose ability to replicate is cell-type dependent. G92A is described in more detail in U.S. application Ser. No. 08/486,147, filed Jun. 7, 1995, the contents of which are incorporated herein by reference.
- In one embodiment of the invention, the immunological properties of the mutated herpes simplex virus consist essentially of inducing an immune response that is specific for the tumor cell type and that kills cells of the inoculated tumor and of a non-inoculated tumor. As used above, the phrase “consisting essentially of” excludes another feature that would affect significantly a material aspect of the invention. For example, in accordance with this embodiment, the genome of the mutated virus does not comprise an expressible immune modulator, such as IL-2. As discussed below, other embodiments of the invention encompass mutant viruses whose genomes do comprise an expressible immune modulator.
- Another embodiment of the present invention relates to a composition, consisting essentially of the herpes simplex virus and a pharmaceutically acceptable carrier, that is administered to a patient who suffers from or who is at risk of developing multiple, metastatic tumors. The composition is administered directly to the tumors cells in situ. In this description, the phrase “consisting essentially of” excludes a step or other feature that would affect significantly a material aspect of the invention. Thus qualified, a composition of this embodiment would include, for example, the prescribed herpes simplex virus with no other virus or defective virus vector; this, because an additional virus would substantially complicate the inventive protocol. The invention also encompasses the administration of this composition in combination with another therapy, such as chemotherapy or radiation treatment.
- In accordance with another embodiment, more than one mutated herpes simplex virus is administered. This embodiment can be effected by administering a single composition comprising more than one mutated herpes simplex virus and a pharmaceutically acceptable vehicle for the viruses, or by, administering more than one composition, each composition comprising at least one mutated herpes simplex virus and a pharmaceutically acceptable vehicle for the virus or viruses. In one embodiment, a composition is administered that comprises (A) a first mutated herpes simplex virus, (B) a second mutated herpes simplex virus and (C) a pharmaceutically acceptable carrier for the viruses. In an another embodiment, a composition is administered that consists essentially of (A) a first mutated herpes simplex virus, (B) a second mutated herpes simplex virus and (C) a pharmaceutically acceptable carrier for the viruses. As set forth above, the phrase “consisting essentially of” excludes a step or other feature that would affect significantly a material aspect of the invention. Thus, this embodiment would entail, for example, the administration of the prescribed first and second herpes simplex viruses with no other virus or defective virus vector.
- The inoculation of a tumor with one or more mutated herpes simplex viruses in accordance with the present invention induces a systemic tumor-specific immune response that is specific for the cell type of the inoculated tumor and that kills cells of the inoculated tumor and of other, non-inoculated tumors. The induced cell death is observed, for example, as inhibited tumor growth or as reduced tumor size. In the examples set forth below, the induced cell death is observed as an inhibition of the growth of the inoculated tumor and of distant, established, non-inoculated tumors. In some instances, the tumors shrink to undetectable sizes. In one of the murine models studied, CT26, the immune response is correlated with cytotoxic T lymphocytes (CD8+) that recognize a major histocompatibility complex (MHC) class I-restricted peptide that is a dominant tumor antigen.
- As discussed above, the composition is administered directly to tumor cells of the patient, in situ. This can be accomplished by procedures known in the art, for example, by intratumoral inoculation during surgery, such as surgery for debulking a tumor, into external melanomas, or stereotactically into the tumor bed. Other approaches for targeting tumors also are appropriate. Generally, the maximum safe dose is administered at weekly intervals if the tumor is readily accessible, or is administered during surgery or tumor biopsy.
- The pharmaceutically acceptable vehicle for the virus can be selected from known pharmaceutically acceptable vehicles, and should be one in which the virus is stable. For example, it can be a diluent, solvent, buffer, and/or preservative. An example of a pharmaceutically acceptable vehicle is phosphate buffer containing NaCl. Other pharmaceutically acceptable vehicles aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 15th Ed. Easton: Mack Publishing Co. pp 1405-1412 and 1461-1487 (1975) and THE NATIONAL FORMULARY XIV., 14th Ed. Washington: American Pharmaceutical Association (1975), the contents of which are hereby incorporated by reference.
- Huang et al., Science 264: 961-65 (1994), demonstrated that the priming of an immune response against a MHC class I-restricted tumor antigen involves the transfer of that antigen to host bone marrow-derived antigen-presenting cells (APCs) prior to its presentation to CD8+ T cells. While not wanting to be bound by any theory, the present inventors believe that local HSV infection of a tumor might induce circulating precursors to differentiate into APCs. A subset of macrophages are able to present exogenous antigens on MHC class I molecules to CD8+ T cell clones. Rock et al., J. Immunol. 150: 438-46 (1993). The lytic destruction or virally-induced death of tumor cells might release tumor antigens which then are picked up by APCs and carried to the draining lymph nodes. There they would be processed and presented to CD8+ T cells. Associative recognition of HSV-specific and tumor-specific antigens might also play a role in the strength of the response. Tumor cells infected with replication-competent HSV would have maturing virions budding from their cell membranes and may also process viral antigens for MHC class-I presentation likes APCs do. The HSV-infected tumor cells therefore might induce T cell-Mediated immune reactions directly. Some of the immune response induced by co-presentation of viral and tumor antigens may be triggered thereafter by only one of the co-expressed antigens.
- In another preferred embodiment, one or more immune modulators are delivered to the tumor cells in addition to the mutated herpes simplex virus described above. Examples of immune modulators useful in the present invention include cytokines, co-stimulatory molecules, and chemokines. Delivery of one or more immune modulators can be effected, for example, by means of a mutated herpes simplex virus that comprises one or more expressible nucleotide sequences encoding one or more cytokines or other immune-modulatory genes, or by means of more than one mutated herpes simplex virus, each of which comprises one or more expressible nucleotide sequences encoding one or more cytokines or other immune-modulatory genes. Non-herpes simplex virus vectors also can be used to effect delivery of one or more immune modulators. For example, one or more adenoviral vectors, adenovirus-associated vectors, retroviral vectors, or vaccinia virus vectors comprising one or more expressible nucleotide sequences encoding one or more immune-modulatory genes can be used in accordance with this embodiment. See, e.g., Shawler et al., Adv. Pharacol. 40: 309-37 (1997), discussing gene transfer of immunostimulatory cytokines.
- The present invention also comprehends a situation where the patient receives both a mutated herpes simplex virus and a defective herpes simplex virus vector which contains the genes for one or more immune modulators, and where the former virus acts as a helper virus for the defective vector. Additionally, the invention encompasses the administration of one or more mutated herpes simplex viruses and more than one defective herpes simplex virus vectors, where each defective vector contains the genes for one or more immune modulators, and where the former virus or viruses act as helpers for the defective vectors. Where one or more helper viruses are administered, the immunological properties of the helper viruses, i.e., the mutated herpes simplex viruses, consist essentially of inducing an immune response that is specific for the tumor cell type and that kills cells of the inoculated tumor and of a non-inoculated tumor. Thus employed, “consisting essentially of” excludes another feature that would affect significantly a material aspect of the invention. Accordingly, the use of this phrase excludes, for example, the administration of a helper virus vector that is capable of expressing an immune modulator, such as IL-2.
- Examples of immune modulators that are useful in accordance with the present invention include IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-12, G-CSF, GM-CSF, IFN-α, IFN-γ, TNF-α and B7. See, e.g., Parmiani et al., Adv. Pharmacol. 40: 259-89 (1997); Shawler et al., Adv. Pharmacol. 40: 309 (1997). For convenience, the use of IL-12 is exemplified in the discussion which follows. It is to be understood, however, that other immune modulators can be used in its place or in addition thereto. Also, where the present description refers to “an immune modulator,” it is to be understood that the invention encompasses one or more immune modulators.
- The cytokine IL-12 is a heterodimeric cytokine, composed of 35 kD (p35) and 40 kD (p40) subunits, that binds to receptors present on NK and T cells. The high-affinity receptor is composed of two β-type cytokine receptor subunits that individually behave as low affinity receptors. IL-12 plays a multi-functional role in the immune system, augmenting the proliferation and cytotoxic activity of T cells and NK cells, regulating IFN-γ production and promoting the development of CD4+ T helper (Th1) cells.
- The antitumor activity of IL-12 has been demonstrated in a number of different murine tumor models, both solid and metastatic, with systemic administration of recombinant IL-12, fibroblasts or tumor cells engineered to secrete IL-12, and viral vectors expressing IL-12. IL-12 immunotherapy is less effective with other tumor cell lines such as CT26, C26, MCH-1-A1, and TS/A. Zitvogel et al., Eur. J. Immunol. 26: 1335-41 (1996). Systemic delivery of rIL-12 has been shown to have potent antitumor effects in various animal models. Prolonged exposure to IL-12 can have deleterious side effects like those observed with many cytokines, however.
- Transfer of immune modulatory genes directly to the tumor cells is advantageous because the genes are expressed within the tumor at the site of their action in concert with putative tumor antigens. In accordance with the present invention, therefore, tumors are modified in situ to make tumor cells a source of immune modulator production.
- Defective herpes simplex virus vectors are plasmid-based vectors which are unable to replicate on their own because they lack viral genes, but which contain specific HSV sequences that, in the presence of helper herpes simplex virus, support DNA replication and subsequent packaging into virus particles. Lim et al., BioTechniques 20(3): 460 (1996); Spaete and Frenkel, Cell 30: 295-304 (1982). In accordance with the present invention, the defective herpes simplex virus vector contains one or more nucleotide sequences encoding one or more cytokines or other immune modulators. Any herpes simplex virus described above can be used as helper virus, such as a replication-competent virus, a replication-defective virus, or a conditionally replication-competent virus. Because a viral genome length of DNA (−153 kb) is packaged, each defective vector can contain multiple copies of the immune modulator gene. For example, a defective vector containing an IL-12 gene can contain approximately 15 copies of the IL-12 gene (based on the size of the IL-12-containing plasmid), which can transduce both dividing and non-dividing cells at high efficiency. The viral DNA does not integrate into the infected cell genome, and with the CMV promoter driving IL-12 expression, expression is strong but transient. In accordance with one aspect of the present invention, a defective. HSV vector is used to deliver one or more immune modulators such as IL-12 in combination with G207 as a helper virus. In accordance with another aspect of the present invention, a defective HSV vector is used to deliver one or more immune modulators such as IL-12 in combination with tsK as a helper virus. The construction of defective herpes virus vectors and their use with helper viruses is known in the art. For example, see Spaete & Frankel, supra, and Geller et al., Proc. Nat'l Acad. Sci. USA 87: 8950-54 (1990).
- The defective IL-12-containing vector infects a number of different tumor cells which then produce and secrete IL-12 in vivo. Cells that are highly susceptible to HSV infection, but where the helper virus replicates poorly and therefore does not rapidly destroy the cells, may be the highest producers of IL-12 in vivo. The IL-12 acts as an adjuvant for the immune response elicited by the herpes simplex virus. In the murine models studied, the enhanced immune response is correlated with heightened induction of tumor-specific CTL activity and IFN-γ production by splenocytes, as described in more detail in the examples below.
- The use of one or more defective herpes simplex virus vectors containing one or more immune modulators and one or more helper herpes simplex viruses in accordance with the present invention kills cells of the inoculated tumor and of other, non-inoculated tumors. This antitumor effect is significantly greater than that observed when a tumor is inoculated with a mutated herpes simplex virus alone, revealing a synergistic effect.
- As discussed above, the immune response elicited in accordance with the present invention kills cells of the inoculated tumor and also kills non-inoculated tumor cells, including cells of distant, non-inoculated tumors. This effect makes this method particularly useful for treating patients presenting with multiple metastatic tumors of a given cell type. It also represents an improvement in the treatment of localized, non-metastatic tumors because the method kills tumor cells that are not directly targeted by the administered virus.
- Any type of tumor can be treated in accordance with the present invention, including non-metastatic tumors, tumors with metastatic potential, and tumors already demonstrating an ability to metastasize. Examples of tumor cell types that can be treated in accordance with the present invention include astrocytoma, oligodendroglioma, meningioma, neurofibroma, glioblastoma, ependymoma, Schwannoma, neurofibrosarcoma, and medulloblastoma cell types. The invention also is useful in treating melanoma cells, pancreatic cancer cells, prostate carcinoma cells, head and neck cancer cells, breast cancer cells, lung cancer cells, colon cancer cells, lymphoma cells, hepatoma cells, ovarian cancer cells, renal cancer cells, neuroblastomas, squamous cell carcinomas, sarcomas, and mesothelioma and epidermoid carcinoma cells.
- The embodiments of the invention are further illustrated through examples which show aspects of the invention in detail. These examples illustrate specific aspects of the invention and do not limit its scope.
- The antitumor efficacy of G207 was evaluated in a bilateral, established subcutaneous tumor model with CT26 cells as described below.
- Cell Line
- The murine colorectal carcinoma CT26 cell line has been widely used as a syngeneic tumor model to study immunotherapy. Fearon et al. Cancer Res. 35: 2975-80 (1988); Wang, et al., J Immunol. 154: 4685-92 (1995); Huang et al., Proc. Natl. Acad. Sci. USA 93: 9730-35 (1996). CT26 is a transplantable colon epithelial tumor induced by intrarectal injections of N-nitroso-N-methylurethane in female BALB/c mice (H-2d). Corbett et al., Cancer Res. 35: 2434-39 (1975).
- In normal mice, CT26 is poorly immunogenic: 103-104 cells can cause a lethal tumor and do not induce detectable tumor-specific CTL. Fearon et al., supra; Wang et al., supra. AH1, a nonmutated nonamer derived from the envelop protein (gp70) of an endogenous ecotropic murine leukemia provirus (MuLV), env-1, has been identified as the immunodominant MHC class I-restricted antigen for CT26. Huang et al., supra. Adoptive transfer of peptide-specific CTL lines has been able to cure established subcutaneous CT26 tumors, demonstrating the correlation between induction of tumor-specific CTL and an antitumor effect.
- Herpes simplex virus does not grow in many rat cells, and attenuated viruses like G207 do not grow well in many mouse tumors either. This is in contrast to their excellent growth in most human tumor lines. However, studies in human tumor lines require the use of athymic mice. CT26 was chosen as a model cell line after several years of trying to find a good syngeneic system for studying the immune effects of attenuated conditionally replicated herpes vectors, such as G207.
- Infection of CT26 Cells
- Tumor cells (1×105) were injected subcutaneously in the bilateral flanks of female BALB/c mice (National Cancer Institute (Rockville, Md.)). When subcutaneous tumors were palpably growing (approximately 5 mm in diameter), mice were unilaterally inoculated into the right side tumor with either G207 virus in 50 μl of virus buffer (150 mM NaCl, 20 mM Tris, pH 7.5) and modified Eagle's medium (MEM) (1:1), or with 50 μl of mock-infected extract (“mock”), prepared from mock-infected cells using the same procedures as those used for the virus inoculum. A second injection of the same composition was given 7 days later in some experiments. Tumor size was measured by external caliper. All animal procedures were approved by the Georgetown University Animal Care and Use Committee.
- As shown in
FIG. 1C , inoculation with G207 resulted in a reduction in tumor growth of both the inoculated tumors (Rt), as well as of their non-inoculated contralateral counterparts (Lt) when compared to mock-inoculated controls (p<0.0005 (Rt) and p<0.001 (Lt) onday 21 postinfection; unpaired t-test). At the time of the second inoculation, 7 days after the first inoculation, lacZ expression from G207 was detected by X-gal histochemistry in the inoculated tumor but not the non-inoculated tumor. - Two intratumoral inoculations with a lower dose of G207 (7×103 plaqueforming units (pfu)) induced significant growth inhibition of the bilateral tumors compared to controls (p<0.01 (Rt) and p<0.05 (Lt) on
day 21 postinfection; unpaired t-test), but to a lesser degree than the higher dose (seeFIG. 1C ). - A single unilateral intratumoral inoculation with 5×107 pfu of G207 caused a large reduction in bilateral tumor growth (
FIG. 1A ), comparable to the double inoculation with 7×105 pfu (FIG. 1C ). - The antitumor effect on the non-inoculated contralateral tumor depended upon intratumoral inoculation of G207, as intradermal inoculation of G207 in the right flanks of mice with established unilateral tumors in the left flanks had no effect on tumor growth (see
FIG. 1B ). - Role of T Cells in Immune Response
- To evaluate the potential role of T cells in the herpes simplex virus-induced inhibition of tumor growth according to the present invention, the antitumor efficacy of intratumoral G207 inoculation was tested in athymic mice. There was no effect of intratumoral inoculation of 7×105 pfu of G207. Higher dose G207 inoculations (5×107 pfu) caused a slight growth inhibition of virus-inoculated tumors compared to mock-inoculated tumors (p=0.08 at day 10), but no effect on non-inoculated contralateral tumors was observed. This lack of effect on contralateral tumors in athymic mice indicates a T cell component to the elicited immune response.
- Tumor-Specific CTL Response
- To determine whether the herpes simplex virus induces a tumor-specific CTL response, effector cells were generated in vitro from splenocytes obtained 12 days after the first virus (G207) inoculation and tested in a 51Cr release assay.
- Single-cell suspensions of splenocytes (3×106) from individual mice treated with G207 or mock were cultured with 1×106 mitomycinC-treated CT26 cells (100 μg/ml of mitomycin C for 1 hr). Effector cells were harvested after 6 days of in vitro culturing and mixed with target cells at the ratios indicated. Target cells were incubated with 50 μCi of Na51CrO4 (51Cr) for 60 min. Four-hour 51Cr release assays were performed as described in Kojima et al., Immunity 1: 357-64 (1994). The % Specific Lysis was calculated from triplicate samples as follows:
-
[(experimental cpm−spontaneous cpm)/(maximum cpm−spontaneous cpm)]×100. - A20 is a B cell lymphoma cell line (Ig+, Ia+, H-2d) derived from a spontaneous reticulum cell neoplasm in BALB/c mice. Kim et al., J. Immunol. 122: 549-54 (1979). It is capable of presenting protein antigen to MHC-restricted antigen-reactive T lymphocytes. Glimcher, et al., J. Exp. Med. 155: 445-59 (1982).
- Mice treated intratumorally with G207 generated a highly specific CTL response against CT26 cells but not against A20 lymphoma cells (also H-2d). No specific CTL response was detected in mice treated intradermally with G207 or intratumorally with mock extract. There was a small non specific CTL response (against A20 and CT26) induced in mock-inoculated mice.
- The ability of CTL generated in mice inoculated intratumorally with the herpes simplex virus to recognize the CT26 immunodominant MHC-class I restricted antigenic peptide AH1 also was evaluated. AH1, the nonamer SPSYVYHQF, is the immunodominant peptide from CT26, presented by the MHC class I Ld molecule. The Ld-binding AH1 peptide is derived from gp70, one of two env gene products of the endogenous MuLV. Huang et al., supra, demonstrated that CT26 cells express the MuLV env gene product while the normal tissues of BALB/c mice do not, and that the viral antigen, gp70, can serve as a potential tumor rejection antigen for the immune system. The AH1 peptide was synthesized by Peptide Technologies (Washington, D.C.) to a purity of >99% as determined by HPLC and amino acid analysis.
- H-2Ld-restricted P815AB.35-43, LPYLGWLVF, is the immunodominant peptide derived from murine mastocytoma P815 cells. Van den Eynde et al., J. Exp. Med. 173: 1373-84 (1991).
- Effector cells from intratumoral G207-inoculated mice exhibited specific lysis of CT26 cells and of A20 cells pulsed with Ld-restricted peptide AH1, but not of A20 cells pulsed with Ld-restricted peptide P815AB. The in vitro CTL activity was completely abrogated by depletion of CD8+ cells, but not by depletion of CD4+ cells.
- Intradermal inoculation with G207 virus or intratumoral inoculation with mock extract did not enhance the activation of specific T cells against CT26 tumors. In contrast, in vivo priming against tumors that express endogenous antigens by intratumoral inoculation of G207 induced an antigenic peptide-specific CTL response. These results indicate that the inoculation of tumors with a herpes simplex virus can overcome potential mechanisms of tolerance to endogenous antigen expression. The lack of an antitumor response, against non-inoculated tumors in athymic mice and the loss of CTL activity by depletion of CD84+ cells in vitro suggests an important role for T cell-mediated, MHC class I-restricted recognition by CTL.
- M3 mouse melanoma cells (3×105) were inoculated bilaterally into the flanks of DBA/2 mice. When the tumors were 5 mm in maximal diameter, the right flank tumor was inoculated one time with either 5×107 pfu of G207 or an equivalent amount of mock Vero cell preparation (as a negative control).
- Inoculation with G207 inhibited the growth of the inoculated tumor (p<0.0005), and also significantly inhibited the growth of the non-inoculated tumor (p<0.02).
FIG. 2 . - Mouse N18 neuroblastoma cells were subcutaneously implanted bilaterally into syngeneic A/J mice. Eight days after tumor implantation, 107 pfu of G207 or mock were injected into the left tumor. In six of eight animals, inoculation with G207 resulted in the disappearance of the tumors on both sides.
FIG. 3 . - N18 neuroblastoma cells were subcutaneously implanted bilaterally into the left flank of A/J mice. Three days later, N18 neuroblastoma cells were intracerebrally implanted into the right frontal lobe of the mice. On
days 10 and 13, the subcutaneous tumors only were injected with G207 (11 mice) or mock (11 mice). Within 35 days of cerebral implantation, all mock-treated mice died from or had intracerebral tumors. Four out of eleven mice treated with G207 had no intracerebral tumors, and one G207-treated mouse was a long-term survivor. G207 treatment inhibited growth of distant, intracerebral tumors and increased the survival of tumor-bearing animals (P<0.05 by Wilcox test). - Rechallenge with N18
- Ten A/J mice with no previous exposure to N18 cells (naive group), thirty A/J mice that had spontaneously rejected prior subcutaneous injections of N18 cells (rejection group) and twelve A/J mice that previously had established N18 subcutaneous tumors that were cured by intratumoral injection of G207 (cured group) were subcutaneously injected with N18 cells. None of the animals of the cured group showed any sign of tumor growth, whereas a large number of animals of the naive and rejection groups showed significant tumor growth.
- Mouse CT26 colon carcinoma cells were subcutaneously implanted bilaterally into syngeneic BALB/c mice. 105 pfu of tsK, a temperature-sensitive herpes simplex virus mutant in ICP4, or mock was injected into the right tumor, and a second inoculation of the same composition was given seven days later (day 7). Inoculation with tsK resulted in significant inhibition of tumor growth in both tumors (p<0.05 on day 21).
FIG. 4 . - The murine colorectal carcinoma cell line CT26 was used to evaluate the antitumor efficacy of a defective vector containing IL-12 and G207 as the helper virus.
- Generation of Defective Vectors
- Two amplicon plasmids of similar size were constructed, pHCIL12-tk and pHCL-tk, which encoded the two subunits of murine IL-12 (p40 and p35) or lacZ, respectively, under control of the CMVIE promoter (see
FIGS. 5A and 5B ). Since IL-12 is functional as a heterodimer, both subunits were expressed from a single defective vector, as a bicistronic message, by means of an internal ribosome entry site (IRES). - The double-cassette amplicon plasmid pHCL-tk was constructed by inserting the HSV-1 thymidine kinase (TK) gene and the blunt-ended BamH1 fragment from pHSV-106 (Life Technologies, Inc., Rockville, Md.) into the blunt-ended Spe I site of pHCL (
FIG. 5A ). - The coding region of p40, BamH1 fragment from BL-pSV40, cDNA for murine. IL-12 p35 and an IRES from equine encephalomyocarditis virus (EMCV) from DFG-mIL-12 (IRES-p35), and BamH1 fragment from DFG-mIL12 were subcloned into LITMUS 28 (New England Biolabs, MA) at the BglII/BamH1 site to generate p40-IRES-35. The IL-12 encoding double-cassette amplicon plasmid pHCIL12-tk was constructed by insertion of the p40-IRES-p35 cassette, SnaB1/AflII fragment, into the blunt-ended SalI site of pSR-ori and then inserting the HSV TK blunt-ended BamH1 fragment into the blunt-ended SphI site to produce pHCIL12-tk.
FIG. 5B . - G207, containing deletions in both copies of the γ34.5 gene and an E. coli lac Z insertion inactivating the ICP6 gene, was used as helper virus for the generation of defective vector (dv) stocks. Vero cells were co-transfected with purified amplicon plasmid DNA (pHCIL12-tk and pHCL-tk) and G207 viral DNA using lipofectAMINE™ (Life Technologies, Inc., Rockville, Md.), as described by the manufacturer, and then cultured at 34.5° C. until they exhibited complete cytopathic effect. Virus was then harvested and passaged at a 1:4 dilution in Vero cells until inhibition of helper virus replication was observed. The IL-12 containing defective vector is called dvIL12/G207 and the lacZ containing defective vector is called dvlacZ/G207.
- Titering of Defective Vector Stocks
- Defective vector stocks were titered after a freeze-thaw/sonication regime and removal of cell debris by low-speed centrifugation (2000×g for 10 min at 4° C.). G207 helper virus titer was expressed as the number of pfus after plaque assay on Vero cells at 34.5° C. For dvIL12/G207, IL-12 expression was determined and the passage with highest level was used (passage 4) with a G207 helper virus titer of 5×107 pfu/ml. The titer of dvlacZ/G207, determined by counting X-gal (5-bromo-4-chloro-3-indolyl-β-D-glactopyranoside) histochemistry positive single cells (defective particle units, dpu) after formation of plaques by G207, was 5×106 dpu/ml and 5×107 pfu/ml of helper virus.
- Cell Culture
- African green monkey kidney (Vero) cells were cultured in DMEM containing 10% calf serum (CS). MC-38 mouse colon adenocarcinoma, Harding-Passey mouse melanoma, MDA-MB-435 human breast adenocarcinoma, and CT26 cells were grown in DMEM containing 10% heat-inactivated FCS (Hyclone, Logan, Utah) and penicillin-streptomycin (Sigma Chemical Co, St Louis, Mo.). A20, a B cell lymphoma cell line (Ig+, Ia+, H-2d) derived from a spontaneous reticulum cell neoplasm in BALB/c mice (American Type Culture Collection, Rockville, Md., ATCC TIB 208) was grown in RPMI 1640 containing 10% heat-inactivated FCS, 50 μM of 2-ME, 2 mM glutamine, 20 mM Hepes buffer, and penicillin-streptomycin.
- Detection of IL-12
- The expression and secretion of IL-12 was determined by ELISA assay after infection of tumor cells in culture at a multiplicity of infection (MOI) of 1 pfu per cell.
- 24 hours post-infection, aliquots of infected cell supernatant were removed, quick frozen in a dry-ice/ethanol bath, and stored at −80° C. for detection of IL-12. Tumors and blood were collected from defective vector-treated mice and snap-frozen in a dry-ice/ethanol bath. Frozen tissue was homogenized in ice-cold PBS containing 500 μM PMSF, 0.5 μg/ml leupeptin and 0.7 μg/ml pepstatin. The homogenate was then sonicated twice for 10 seconds and cleared by centrifugation in a microfuge for 5 min at 4° C. Immunoreactive IL-12 levels were determined by sandwich ELISA, using Ab pairs and rIL-12. The rIL-12 standards were diluted in the same media or buffer as the samples (i.e., mouse serum for the serum samples).
- Briefly, 96-well plates coated with an anti-mouse IL-12 mAb (9A5) were incubated overnight at room temperature with the test samples. After washes, the plates were incubated with peroxidase-labelled anti-mouse IL-12 p40 Ab (5C3) for 2 hours and then were developed. Absorbance was measured at 450 nm.
- Infection of CT26 (murine colon carcinoma), Harding-Passey (murine melanoma), MCA38 (murine colon adenocarcinoma) and MDA-MB-435 (human breast adenocarcinoma) cells with dvIL12/G207 resulted in secretion of up to 1.5 ng murine IL-12/105 tumor cells in 24 hours.
FIG. 6 . No IL-12 was detected in the supernatants of uninfected tumor cell cultures or those infected with dvlacZ/G207. Levels of IL-12 synthesis and secretion peaked 1 day after dvIL12/G207 infection of CT26 cells and decreased to undetectable levels by 3 days post-infection, likely due to cell death. - Subcutaneous Tumor Model
- BALB/c and BALB/c (nu/nu) mice were obtained from the National Cancer Institute or Charles River (Wilmington, Mass.). All animal procedures were approved by the Georgetown University Animal Care and Use Committee.
- CT26 tumor cells (1×105) were injected subcutaneously (s.c.) in the bilateral flanks of mice. When s.c. tumors were palpably growing (approximately 5 mm in maximal diameter), mice were unilaterally inoculated into the right side tumor with either 50 μl of defective HSV vector (7×105 pfu of helper virus) in virus buffer (150 mM NaCl, 20 mM Tris, pH 7.5) or 50 μl virus buffer, followed by a second injection of the
same composition 7 days later. Where indicated, mock extract was used in place of virus buffer. DvlacZ/G207 rather than helper virus G207 alone was used as a control for dvIL12/G207 inoculation so that differences in viral factors (i.e., particle:pfu ratio) present in defective vector stocks versus G207 stocks would be accounted for. Both G207 and dvlacZ contain E. coli lacZ and therefore no additional foreign antigens were expressed by the control defective vector. - Tumor size was measured by external caliper and tumor volume was calculated (V=h×w×d). If animals appeared moribund or the diameter of their s.c. tumors reached 18 mm, they were sacrificed and this was recorded as the date of death for survival studies. Statistical differences were calculated using StatView 4.5 (Abacus Concepts Inc., Berkeley, Calif.) where mean tumor volume was assessed by unpaired t-test, survival means by ANOVA (Fisher's post-hoc comparisons) and differences in survival by Logrank (Mantel-Cox) test.
- Inoculation with dvIL12/G207 elicited a very prominent antitumor effect, with both the inoculated tumors as well as their non-inoculated contralateral counterparts demonstrating a significant reduction in tumor growth.
FIG. 7 . Two out of six of the dvIL12/G207 inoculated tumors regressed to an undetectable size. Inoculation with dvlacZ/G207 also resulted in a significant reduction in tumor growth of both inoculated and non-inoculated tumors compared to controls, although to a much lesser extent than dvIL12/G207.FIG. 7 . - Mice also were followed for survival, where sacrifice occurred when either of the bilateral tumors became larger than 18 mm in diameter. Survival of the defective vector-treated animals is therefore reflective of the growth of the non-inoculated tumors and was significantly longer than control animals. Mice treated unilaterally with dvIL12/G207 survived longer than dvlacZ/G207 treated mice (
FIG. 8 ). IL-12 was detected in the dvIL12/G207 inoculated tumors one and five days post-inoculation (approximately 50-100 pg/tumor), with no IL-12 detected in the serum. - Role of T Cells in Immune Response
- To evaluate the possible role of T cells in the defective HSV vector-induced antitumor response, bilateral CT26 s.c. tumors were established in athymic BALB/c (nu/nu) mice. As with the immune-competent murine model discussed above, unilateral intratumoral inoculation of dvIL12/G207, dvlacZ/G207 or mock-extract was performed into the right side tumors when they were palpable (approximately 5 mm in maximal diameter), and a second inoculation of the same composition was given seven days later.
- Although there was a slight delay in growth of right side tumors injected with dvIL12/G207, no significant tumor growth inhibition was observed in either the inoculated or contralateral non-inoculated tumors. CT26 tumors grew somewhat more rapidly in the athymic mice than in the immune-competent mice.
- Tumor-Specific CTL Response
- To test whether inhibition of tumor growth was associated with increased CTL activity, the ability of intratumoral inoculation with defective HSV vectors to elicit CT26-specific CTL activity in vitro was examined using a 51Cr release assay.
- BALB/c mice were inoculated with dvIL12/G207 or dvlacZ/G207 intratumorally when s.c. tumors reached approximately 5 mm in maximal diameter, and a second inoculation of the same composition was given seven days later. Single-cell suspensions of splenocytes were cultured in RPMI 1640 medium with 10% inactivated FCS, 50 μM 2-ME, 2 mM glutamine, 20 mM Hepes, and penicillin-streptomycin in 24-well plates at a concentration of 3×106 cells/ml. In addition, either 1×106 inactivated CT26 cells or 1 μg/ml of peptide AH1 was added to the medium. For inactivation, CT26 tumor cells were incubated for 1 hour in culture medium containing 100 μg/ml of mitomycin C and then washed 2 times. Effector cells were harvested after 6 days of in vitro culture.
- Four-hour 51Cr release assays were performed as described above. In brief, target cells were incubated with 50 μCi of Na51CrO4 (51Cr) for 60 min. A20 cells were pulsed with 1 μg/ml of the Ld-restricted peptides AH1 or P815AB for 1 h before labeling. Target cells were then mixed with effector cells for 4 h at the E/T ratios indicated. The amount of 51Cr release was determined by γ counting, and the percent specific lysis was calculated from triplicate samples as follows:
-
[(experimental cpm−spontaneous cpm)/(maximum cpm−spontaneous cpm)]×100. - Effector cells from dvIL12/G207 treated mice restimulated with mitomycin-C treated CT26 cells exhibited specific lysis of CT26 target cells and of A20 cells pulsed with peptide AH1. No apparent lysis of unpulsed A20 cells or A20 cells pulsed with Ld-restricted peptide P815AB was observed. Effector cells restimulated with peptide AH1 from mice treated with dvIL12/G207 or dvlacZ/G207 exhibited specific lysis of target A20 cells pulsed with peptide AH1 and of CT26 cells, but not of unpulsed A20 cells. The level of CTL activity generated by dvIL12/G207 was significantly greater than that generated by dvlacZ/G207. Effector cells from dvIL12/G207 inoculated animals, not restimulated, were able to specifically lyse CT26 but not A20 cells.
- The effect of intratumoral IL-12 expression on the accumulation of particular T lymphocyte subtypes or IFN-γ production also was determined. Splenocytes were isolated five days after the second inoculation of dvIL12/G207 or dvlacZ/G207 and tested for IFN-γ production by ELISA and splenic T lymphocyte subsets by FACS analysis. Briefly, single-cell suspensions of splenocytes were washed and resuspended in RPMI 1640 medium containing 10% inactivated FCS. Cells (3×106/ml) were cultured in 24-well plates for 24 h. Supernatants were collected and assayed by a sandwich ELISA using anti-IFN-γ Ab pairs obtained from Endogen (Woburn, Mass.).
- Similar percentages of a helper T cells (CD4) and a cytotoxic T cells (CD8a) were found in dvIL12/G207 and dvlacZ/G207 treated mice. Splenocytes from mice treated with dvIL12/G207 produced significantly greater amounts of IFN-γ than those treated with dvlacZ/G207, as shown below.
-
Treatment IFN-γ (ng/ml) dvIL12 16 ± 6 dvlacZ 1.6 ± 0.6 - Defective vectors containing IL-12 and tsK or lacZ and tsK were prepared. Defective vector plasmids pHCIL12-tk and pHCL-tk were prepared as described above. Defective vectors were generated by co-transfection of Vero cells with helper virus tsK DNA and pHCIL12-tk or pHCL-tk. Transfected cells were incubated at 31.5° C. (a replication-permissive temperature for tsK) until total cytopathic effect was observed. The cells then were passaged as described above for G207 helper virus. See also Kaplitt et al., Mol. Cell. Neurosci. 2: 320-30 (1991). The defective vector containing IL-12 is called dvIL12/tsK and the defective vector containing lacZ is called dvlacZ/tsK.
- CT26 mouse colon carcinoma cells were subcutaneously implanted bilaterally into syngeneic BALB/c mice, as described above. The right tumor was inoculated with either dvlacZ/tsK, dvIL12/tsK or mock, and a second inoculation of the same composition was given seven days later. Inoculation with dvlacZ/tsK resulted in a significant inhibition of tumor growth in both tumors (p<0.01 on day 22). Inoculation with dvIL12/tsK resulted in greater inhibition of tumor growth in both tumors compared to dvlacZ/tsK-inoculated tumor (p<0.001).
FIG. 9 . - The survival of inoculated mice also was followed. Mice were sacrificed when they became moribund or when their tumors reached greater than 18 mm in diameter. As shown in
FIG. 10 , mice inoculated with dvlacZ survived significantly longer than mice inoculated with mock (p<0.01), and mice inoculated with dvIL12/tsK survived significantly longer than mice inoculated with dvlac/tsK or mock (p<0.01). - Harding-Passey melanoma cells were subcutaneously implanted into the bilateral flanks of C57BL/6 mice. When the tumors were about 5 mm in maximal diameter (day 0), the right flank tumors were injected with defective vector dvlacZ/tsk (generated from the amplicon plasmid pHCL-tk and expressing E. coil lacZ) or dvGMCSF/tsK (generated from the amplicon plasmid pHCGMCSF-tk, whose structure is the same as pHCIL12-tk except it contains mouse GM-CSF cDNA in place of IL-12 DNA; expression of GM-CSF was detected by ELISA) and helper tsK virus, or with virus buffer. Mice treated with dvGMCSF/tsK showed increased survival over mice treated with dvlacZ/tsk or buffer, and showed decreased tumor growth in both bilateral tumors.
- It is to be understood that the description, specific examples and data, while indicating exemplary embodiments, are given by way of illustration and are not intended to limit the present invention. Various changes and modifications within the present invention will become apparent to the skilled artisan from the discussion, disclosure and data contained herein, and thus are considered part of the invention.
Claims (34)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US13/769,182 US9827307B2 (en) | 1997-08-12 | 2013-02-15 | Use of herpes vectors for tumor therapy |
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US5514297P | 1997-08-12 | 1997-08-12 | |
US09/064,174 US6379674B1 (en) | 1997-08-12 | 1998-04-22 | Use of herpes vectors for tumor therapy |
US10/079,534 US20020127246A1 (en) | 1997-08-12 | 2002-02-22 | Use of herpes vectors for tumor therapy |
US11/097,391 US20050232907A1 (en) | 1997-08-12 | 2005-04-04 | Use of herpes vectors for tumor therapy |
US13/769,182 US9827307B2 (en) | 1997-08-12 | 2013-02-15 | Use of herpes vectors for tumor therapy |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/097,391 Continuation US20050232907A1 (en) | 1997-08-12 | 2005-04-04 | Use of herpes vectors for tumor therapy |
Publications (2)
Publication Number | Publication Date |
---|---|
US20130224243A1 true US20130224243A1 (en) | 2013-08-29 |
US9827307B2 US9827307B2 (en) | 2017-11-28 |
Family
ID=26733894
Family Applications (5)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/064,174 Expired - Fee Related US6379674B1 (en) | 1997-08-12 | 1998-04-22 | Use of herpes vectors for tumor therapy |
US10/079,534 Abandoned US20020127246A1 (en) | 1997-08-12 | 2002-02-22 | Use of herpes vectors for tumor therapy |
US11/097,391 Abandoned US20050232907A1 (en) | 1997-08-12 | 2005-04-04 | Use of herpes vectors for tumor therapy |
US12/194,872 Expired - Fee Related US8361978B2 (en) | 1997-08-12 | 2008-08-20 | Use of herpes vectors for tumor therapy |
US13/769,182 Expired - Fee Related US9827307B2 (en) | 1997-08-12 | 2013-02-15 | Use of herpes vectors for tumor therapy |
Family Applications Before (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/064,174 Expired - Fee Related US6379674B1 (en) | 1997-08-12 | 1998-04-22 | Use of herpes vectors for tumor therapy |
US10/079,534 Abandoned US20020127246A1 (en) | 1997-08-12 | 2002-02-22 | Use of herpes vectors for tumor therapy |
US11/097,391 Abandoned US20050232907A1 (en) | 1997-08-12 | 2005-04-04 | Use of herpes vectors for tumor therapy |
US12/194,872 Expired - Fee Related US8361978B2 (en) | 1997-08-12 | 2008-08-20 | Use of herpes vectors for tumor therapy |
Country Status (12)
Country | Link |
---|---|
US (5) | US6379674B1 (en) |
EP (2) | EP1003533B1 (en) |
JP (2) | JP5078195B2 (en) |
AT (1) | ATE342724T1 (en) |
AU (1) | AU8824998A (en) |
CA (1) | CA2301327C (en) |
DE (1) | DE69836219T2 (en) |
ES (2) | ES2627764T3 (en) |
HK (1) | HK1028560A1 (en) |
LU (1) | LUC00032I2 (en) |
PT (1) | PT1776957T (en) |
WO (1) | WO1999007394A1 (en) |
Families Citing this family (39)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6379674B1 (en) * | 1997-08-12 | 2002-04-30 | Georgetown University | Use of herpes vectors for tumor therapy |
WO2000040734A1 (en) * | 1998-12-31 | 2000-07-13 | Arch Development Corporation | Recombinant herpes simplex virus useful for treating neoplastic disease |
JP2003501445A (en) * | 1999-06-08 | 2003-01-14 | ユーエイビー リサーチ ファンデーション | Herpes simplex virus expressing foreign gene and method of treating cancer with the same |
US6764675B1 (en) | 1999-06-08 | 2004-07-20 | The Uab Research Foundation | Herpes simplex virus expressing foreign genes and method for treating cancers therewith |
AU2584901A (en) * | 1999-12-22 | 2001-07-03 | Onyx Pharmaceuticals, Inc. | Herpes simplex virus-1 glycoprotein c mutants for treating unwanted hyperproliferative cell growth |
US7223593B2 (en) | 2000-01-21 | 2007-05-29 | Biovex Limited | Herpes virus strains for gene therapy |
ATE508635T1 (en) | 2001-03-27 | 2011-05-15 | Catherex Inc | VIRUS VECTORS AND THEIR USE IN THERAPEUTIC METHODS |
WO2002087625A1 (en) * | 2001-05-02 | 2002-11-07 | Ramot At Tel-Aviv University Ltd. | Composite oncolytic herpes virus vectors |
DE10131148A1 (en) * | 2001-06-28 | 2003-01-16 | I P L Internat Pharmaceutics L | Xenogenic oligo- and / or polyribonucleotides as agents for the treatment of malignant tumors |
JP2005519091A (en) * | 2002-03-01 | 2005-06-30 | スローン − ケタリング・インスティテュート・フォー・キャンサー・リサーチ | Prevention of cancer recurrence and metastasis |
US8216564B2 (en) * | 2002-05-02 | 2012-07-10 | Ramot At Tel-Aviv University Ltd. | Composite oncolytic herpes virus vectors |
JP2004099584A (en) * | 2002-05-02 | 2004-04-02 | Keio Gijuku | Antitumor agent using hsv |
ES2463442T3 (en) | 2004-03-31 | 2014-05-28 | Tomoki Todo | Enhancer of anti-cancer activity in viral therapy and method of prevention or treatment of cancer |
WO2006002394A2 (en) | 2004-06-24 | 2006-01-05 | New York University | Avirulent oncolytic herpes simplex virus strains engineered to counter the innate host response |
US8450106B2 (en) * | 2007-10-17 | 2013-05-28 | The Ohio State University Research Foundation | Oncolytic virus |
WO2009052426A2 (en) * | 2007-10-17 | 2009-04-23 | The Ohio State University | Oncolytic virus |
KR100905419B1 (en) | 2008-09-11 | 2009-07-02 | 연세대학교 산학협력단 | Uses of sesquiterpene derivatives |
EP2687609B1 (en) | 2008-11-10 | 2017-01-04 | The United States of America, as represented by The Secretary, Department of Health and Human Services | Method for treating solid tumor |
US9150926B2 (en) | 2010-12-06 | 2015-10-06 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Diagnosis and treatment of adrenocortical tumors using human microRNA-483 |
CA2846372C (en) | 2011-09-08 | 2021-01-12 | New York University | Oncolytic herpes simplex virus and therapeutic uses thereof |
AU2015362687B2 (en) | 2014-12-18 | 2021-12-09 | Amgen Inc. | Stable frozen herpes simplex virus formulation |
US10967015B2 (en) | 2015-06-15 | 2021-04-06 | New York University | Method of treatment using oncolytic viruses |
EP3217993A1 (en) | 2015-07-20 | 2017-09-20 | Virttu Biologics Limited | Treatment of cancer by infusion of oncolytic herpes simplex virus to the blood |
EP3805376A1 (en) | 2016-01-08 | 2021-04-14 | Replimune Limited | Engineered virus |
LT3480307T (en) * | 2016-05-30 | 2021-09-27 | Astellas Pharma Inc. | New genetically-modified vaccinia virus |
US10888594B2 (en) | 2016-05-30 | 2021-01-12 | National University Corporation Tottori University | Genetically engineered vaccinia viruses |
US20190336552A1 (en) | 2016-05-30 | 2019-11-07 | Astellas Pharma Inc. | Genetically engineered vaccinia viruses |
WO2018026872A1 (en) * | 2016-08-01 | 2018-02-08 | Virogin Biotech Canada Ltd | Oncolytic herpes simplex virus vectors expressing immune system-stimulatory molecules |
US20200009204A1 (en) | 2017-03-15 | 2020-01-09 | Amgen Inc. | Use of oncolytic viruses, alone or in combination with a checkpoint inhibitor, for the treatment of cancer |
DK3661954T3 (en) | 2017-08-03 | 2022-04-19 | Amgen Inc | INTERLEUKIN-21 MUTEINS AND METHODS OF TREATMENT |
AU2018314227A1 (en) | 2017-08-07 | 2020-02-06 | Amgen Inc. | Treatment of triple negative breast cancer or colorectal cancer with liver metastases with an anti PD-L1 antibody and an oncolytic virus |
EP3679040B1 (en) | 2017-09-08 | 2022-08-03 | Amgen Inc. | Inhibitors of kras g12c and methods of using the same |
KR20200110358A (en) | 2018-01-12 | 2020-09-23 | 암젠 인크 | Anti-PD-1 Antibodies and Methods of Treatment |
CN108635380A (en) * | 2018-04-13 | 2018-10-12 | 北京唯源立康生物科技有限公司 | Recombination oncolytic virus composition and its application in preparing the drug for treating tumour |
CN112739360A (en) | 2018-09-26 | 2021-04-30 | 安斯泰来制药株式会社 | Cancer therapy based on a combination of oncolytic vaccinia virus and immune checkpoint inhibitors, and pharmaceutical compositions and combination medicaments for use therein |
MX2021007639A (en) | 2018-12-27 | 2021-08-11 | Amgen Inc | Lyophilized virus formulations. |
JP2022522817A (en) | 2019-03-05 | 2022-04-20 | アムジエン・インコーポレーテツド | Use of oncolytic virus to treat cancer |
TW202102543A (en) | 2019-03-29 | 2021-01-16 | 美商安進公司 | Use of oncolytic viruses in the neoadjuvant therapy of cancer |
US11779660B2 (en) * | 2021-04-02 | 2023-10-10 | Krystal Biotech, Inc. | Viral vectors for cancer therapy |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8361978B2 (en) * | 1997-08-12 | 2013-01-29 | Georgetown University | Use of herpes vectors for tumor therapy |
US8871193B2 (en) * | 2001-03-27 | 2014-10-28 | Catherex, Inc. | Viral vectors and their use in therapeutic methods |
Family Cites Families (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
NL9001828A (en) | 1990-08-15 | 1992-03-02 | Centraal Diergeneeskundig Inst | METHOD FOR CHANGING THE CELL, TISSUE, OR HOST TRPISM OF A MICRO-ORGANISM; SO OBTAINED RECOMBINED MICROORGANISM AND ITS APPLICATION IN MEDICINE AND VETERINARY. |
WO1992014821A2 (en) | 1990-10-25 | 1992-09-03 | Massachusetts Institute Of Technology | Nestin expression as an indicator of neuroepithelial tumors |
EP0675961B1 (en) * | 1992-03-31 | 2002-11-27 | Arch Development Corporation | Treatment of tumorigenic disease with a modified HSV |
US5639656A (en) * | 1994-03-31 | 1997-06-17 | Medical College Of Hampton Road | Antibodies reactive with biological markers of benign prostate hyperplasia |
US5571515A (en) | 1994-04-18 | 1996-11-05 | The Wistar Institute Of Anatomy & Biology | Compositions and methods for use of IL-12 as an adjuvant |
US6699468B1 (en) * | 1994-06-23 | 2004-03-02 | Georgetown University | Replication-competent herpes simplex virus mediates destruction of neoplastic cells |
US5585096A (en) | 1994-06-23 | 1996-12-17 | Georgetown University | Replication-competent herpes simplex virus mediates destruction of neoplastic cells |
US5728379A (en) | 1994-06-23 | 1998-03-17 | Georgetown University | Tumor- or cell-specific herpes simplex virus replication |
JP3822261B2 (en) * | 1994-09-09 | 2006-09-13 | 財団法人癌研究会 | Cancer gene therapy |
GB9511101D0 (en) | 1995-06-01 | 1995-07-26 | British Tech Group | Magnetic coil |
WO1997026904A1 (en) * | 1996-01-25 | 1997-07-31 | Medical Research Council | Treatment of non-neuronal cancer using hsv mutant |
US6051428A (en) * | 1997-03-21 | 2000-04-18 | Sloan-Kettering Institute For Cancer Research | Rapid production of autologous tumor vaccines |
-
1998
- 1998-04-22 US US09/064,174 patent/US6379674B1/en not_active Expired - Fee Related
- 1998-08-12 EP EP98939886A patent/EP1003533B1/en not_active Revoked
- 1998-08-12 EP EP06021697.5A patent/EP1776957B1/en not_active Expired - Lifetime
- 1998-08-12 JP JP2000506984A patent/JP5078195B2/en not_active Expired - Lifetime
- 1998-08-12 PT PT60216975T patent/PT1776957T/en unknown
- 1998-08-12 WO PCT/US1998/016447 patent/WO1999007394A1/en active IP Right Grant
- 1998-08-12 DE DE69836219T patent/DE69836219T2/en not_active Expired - Lifetime
- 1998-08-12 ES ES06021697.5T patent/ES2627764T3/en not_active Expired - Lifetime
- 1998-08-12 AU AU88249/98A patent/AU8824998A/en not_active Abandoned
- 1998-08-12 AT AT98939886T patent/ATE342724T1/en active
- 1998-08-12 ES ES98939886T patent/ES2276470T3/en not_active Expired - Lifetime
- 1998-08-12 CA CA2301327A patent/CA2301327C/en not_active Expired - Lifetime
-
2000
- 2000-11-30 HK HK00107700A patent/HK1028560A1/en not_active IP Right Cessation
-
2002
- 2002-02-22 US US10/079,534 patent/US20020127246A1/en not_active Abandoned
-
2005
- 2005-04-04 US US11/097,391 patent/US20050232907A1/en not_active Abandoned
-
2008
- 2008-08-20 US US12/194,872 patent/US8361978B2/en not_active Expired - Fee Related
-
2011
- 2011-11-04 JP JP2011242601A patent/JP2012067114A/en active Pending
-
2013
- 2013-02-15 US US13/769,182 patent/US9827307B2/en not_active Expired - Fee Related
-
2017
- 2017-08-09 LU LU00032C patent/LUC00032I2/fr unknown
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8361978B2 (en) * | 1997-08-12 | 2013-01-29 | Georgetown University | Use of herpes vectors for tumor therapy |
US8871193B2 (en) * | 2001-03-27 | 2014-10-28 | Catherex, Inc. | Viral vectors and their use in therapeutic methods |
US9555072B2 (en) * | 2001-03-27 | 2017-01-31 | The General Hospital Corporation | Viral vectors and their use in therapeutic methods |
Also Published As
Publication number | Publication date |
---|---|
LUC00032I2 (en) | 2017-10-11 |
HK1028560A1 (en) | 2001-02-23 |
US20020127246A1 (en) | 2002-09-12 |
PT1776957T (en) | 2017-06-12 |
ES2276470T3 (en) | 2007-06-16 |
EP1776957B1 (en) | 2017-03-01 |
DE69836219D1 (en) | 2006-11-30 |
DE69836219T2 (en) | 2007-08-23 |
JP2001513508A (en) | 2001-09-04 |
ATE342724T1 (en) | 2006-11-15 |
EP1776957A3 (en) | 2009-06-24 |
US6379674B1 (en) | 2002-04-30 |
JP5078195B2 (en) | 2012-11-21 |
ES2627764T3 (en) | 2017-07-31 |
CA2301327A1 (en) | 1999-02-18 |
CA2301327C (en) | 2011-06-14 |
JP2012067114A (en) | 2012-04-05 |
EP1776957A2 (en) | 2007-04-25 |
US20050232907A1 (en) | 2005-10-20 |
US20090053178A1 (en) | 2009-02-26 |
AU8824998A (en) | 1999-03-01 |
EP1003533A1 (en) | 2000-05-31 |
WO1999007394A1 (en) | 1999-02-18 |
US8361978B2 (en) | 2013-01-29 |
EP1003533B1 (en) | 2006-10-18 |
US9827307B2 (en) | 2017-11-28 |
LUC00032I1 (en) | 2017-08-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US9827307B2 (en) | Use of herpes vectors for tumor therapy | |
US20200345835A1 (en) | Viral vectors and their use in therapeutic methods | |
Herrlinger et al. | Pre-existing herpes simplex virus 1 (HSV-1) immunity decreases, but does not abolish, gene transfer to experimental brain tumors by a HSV-1 vector | |
AU2002306919A1 (en) | Viral vectors and their use in therapeutic methods | |
US6774119B1 (en) | Herpes simplex virus type 1 (hsv-1)-derived vector for selectively inhibiting malignant cells and methods for its use to treat cancers and to express desired traits in malignant and non-malignant mammalian cells | |
JP2003501445A (en) | Herpes simplex virus expressing foreign gene and method of treating cancer with the same | |
MXPA02002142A (en) | Cell specific and or tumor specific promoter retargeting of herpes gamma 34.5 gene expression. | |
Kanno et al. | Experimental gene therapy against subcutaneously implanted glioma with a herpes simplex virus-defective vector expressing interferon-γ |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STCF | Information on status: patent grant |
Free format text: PATENTED CASE |
|
FEPP | Fee payment procedure |
Free format text: MAINTENANCE FEE REMINDER MAILED (ORIGINAL EVENT CODE: REM.); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY |
|
LAPS | Lapse for failure to pay maintenance fees |
Free format text: PATENT EXPIRED FOR FAILURE TO PAY MAINTENANCE FEES (ORIGINAL EVENT CODE: EXP.); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY |
|
STCH | Information on status: patent discontinuation |
Free format text: PATENT EXPIRED DUE TO NONPAYMENT OF MAINTENANCE FEES UNDER 37 CFR 1.362 |
|
FP | Lapsed due to failure to pay maintenance fee |
Effective date: 20211128 |
|
AS | Assignment |
Owner name: NIH - DEITR, MARYLAND Free format text: CONFIRMATORY LICENSE;ASSIGNOR:GEORGETOWN UNIVERSITY;REEL/FRAME:065618/0398 Effective date: 20231108 |